Tumor-targeted agonistic cd28 antigen binding molecules

ABSTRACT

The present invention relates to tumor targeted bispecific agonistic antigen binding molecules characterized by monovalent binding to CD28, methods for their production, pharmaceutical compositions containing these antibodies, and methods of using the same.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 16, 2019, is namedP35128-US_SeqListing.txt and is 958,930 bytes in size and updated by afile entitled P35128_SeqListing_Replacement.txt, created on Aug. 1,2022, which is 961,079 bytes in size.

FIELD OF THE INVENTION

The present invention relates to tumor-targeted bispecific agonisticCD28 antigen binding molecules characterized by monovalent binding toCD28, methods for their production, pharmaceutical compositionscontaining these molecules, and their use as immunomodulators in thetreatment of cancer.

BACKGROUND

Cancer immunotherapy is becoming an increasingly effective therapyoption that can result in dramatic and durable responses in cancer typessuch as melanoma, non-small cell lung cancer and renal cell carcinoma.This is mostly driven by the success of several immune checkpointblockades including anti-PD-1 (e.g. Keytruda, Merck; Opdivo, BMS),anti-CTLA-4 (e.g. Yervoy, BMS) and anti-PD-L1 (e.g. Tecentriq, Roche).These agents are likely to serve as standard of care for many cancertypes, or as the backbone of combination therapies, however, only afraction of patients (<25%) benefits from such therapies. Furthermore,various cancers (prostate cancer, colorectal cancer, pancreatic cancer,sarcomas, non-triple negative breast cancer etc.) present primaryresistance to these immunomodulators. A number of reports indicate thatthe absence of pre-existing anti-tumor T cells contributes to theabsence or poor response of some patients. In summary, despiteimpressive anti-cancer effects of existing immunotherapies, there is aclear medical need for addressing a large cancer patient population andfor developing therapies that aim to induce and enhance noveltumor-specific T cell responses.

CD28 is the founding member of a subfamily of costimulatory moleculescharacterized by paired V-set immunoglobulin superfamily (IgSF) domainsattached to single transmembrane domains and cytoplasmic domains thatcontain critical signaling motifs (Carreno and Collins, 2002). Othermembers of the subfamily include ICOS, CTLA-4, PD1, PD1H, TIGIT, andBTLA (Chen and Flies, 2013). CD28 expression is restricted to T cellsand prevalent on all naïve and a majority of antigen-experiencedsubsets, including those that express PD-1 or CTLA-4. CD28 and CTLA-4are highly homologous and compete for binding to the same B7 moleculesCD80 and CD86, which are expressed on dendritic cells, B cells,macrophages, and tumor cells (Linsley et al., 1990). The higher affinityof CTLA-4 for the B7 family of ligands allows CTLA-4 to outcompete CD28for ligand binding and suppress effector T cells responses (Engelhardtet al., 2006). In contrast, PD-1 was shown to inhibit CD28 signaling byin part dephosphorylating the cytoplasmic domain of CD28 (Hui et al.,2017). Ligation of CD28 by CD80 or CD86 on the surface of professionalantigen-presenting cells is strictly required for functional de novopriming of naïve T cells, subsequent clonal expansion, cytokineproduction, target cell lysis, and formation of long-lived memory.Binding of CD28 ligands also promotes the expression of inducibleco-stimulatory receptors such as OX-40, ICOS, and 4-1BB (reviewed inAcuto and Michel, 2003). Upon ligation of CD28, a disulfide-linkedhomodimer, the membrane proximal YMNM motif and the distal PYAP motifhave been shown to complex with several kinases and adaptor proteins(Boomer and Green, 2010). These motifs are important for the inductionof IL2 transcription, which is mediated by the CD28-dependent activationof NFAT, AP-1, and NFκB family transcription factors (Fraser et al.,1991) (June et al., 1987) (Thompson et al., 1989). However, additionalpoorly characterized sites for phosphorylation and ubiquitination arefound within the cytoplasmic domain of CD28. As reviewed by (Esensten etal., 2016), CD28-initiated pathways have critical roles in promoting theproliferation and effector function of conventional T cells. CD28ligation also promotes the anti-inflammatory function of regulatory Tcells. CD28 co-stimulates T cells by in part augmenting signals from theT cell receptor, but was also shown to mediate unique signaling events(Acuto and Michel, 2003; Boomer and Green, 2010; June et al., 1987).Signals specifically triggered by CD28 control many important aspects ofT cell function, including phosphorylation and other post-translationalmodifications of downstream proteins (e.g., PI3K mediatedphosphorylation), transcriptional changes (eg. Bc1-xL expression),epigenetic changes (e.g. IL-2 promoter), cytoskeletal remodeling (e.g.orientation of the microtubule-organizing center) and changes in theglycolytic rate (e.g. glycolytic flux). CD28-deficient mice have reducedresponses to infectious pathogens, allograft antigens, graft-versus-hostdisease, contact hypersensitivity and asthma (Acuto and Michel, 2003).Lack of CD28-mediated co-stimulation results in reduced T cellproliferation in vitro and in vivo, in severe inhibition ofgerminal-centre formation and immunoglobulin isotype-class switching,reduced T helper (Th)-cell differentiation and the expression ofTh2-type cytokines. CD4-dependent cytotoxic CD8+ T-cell responses arealso affected. Importantly, CD28-deficient naïve T cells showed areduced proliferative response particularly at lower antigenconcentrations. A growing body of literature supports the idea thatengaging CD28 on T cells has anti-tumor potential. Recent evidencedemonstrates that the anti-cancer effects of PD-L1/PD-1 and CTLA-4checkpoint inhibitors depend on CD28 (Kamphorst et al., 2017; Tai etal., 2007). Clinical studies investigating the therapeutic effects ofCTLA-4 and PD-1 blockade have shown exceptionally promising results inpatients with advanced melanoma and other cancers. In addition, infusionof genetically engineered T cells expressing artificial chimeric T cellreceptors comprising an extracellular antigen recognition domain fusedto the intracellular TCR signaling domains (CD3z) and intracellularco-stimulatory domains (CD28 and/or 4-1BB domains) has shown high ratesand durability of response in B cell cancers and other cancers.

CD28 agonistic antibodies can be divided into two categories: (i) CD28superagonistic antibodies and (ii) CD28 conventional agonisticantibodies. Normally, for the activation of naïve T cells bothengagement of the T cell antigen receptor (TCR, signal 1) andcostimulatory signaling by CD28 (signal 2) is required. CD28Superagonists (CD28SA) are CD28-specific monoclonal antibodies, whichare able to autonomously activate T cells without overt T cell receptorengagement (Hung, 2012). In rodents, CD28SA activates conventional andregulatory T cells. CD28SA antibodies are therapeutically effective inmultiple models of autoimmunity, inflammation and transplantation.However, a phase I study of the human CD28SA antibody TGN1412 resultedin a life-threatening cytokine storm in 2006. Follow-up studies havesuggested that the toxicity was caused by dosing errors due todifferences in the CD28 responsiveness of human T cells and T cells ofpreclinical animal models. TGN1412 is currently being re-evaluated in anopen-label, multi-center dose escalation study in RA patients andpatients with metastatic or unresectable advanced solid malignancies.CD28 conventional agonistic antibodies, such as clone 9.3, mimic CD28natural ligands and are only able to enhance T cell activation inpresence of a T cell receptor signal (signal 1). Published insightsindicate that the binding epitope of the antibody has a major impact onwhether the agonistic antibody is a superagonist or a conventionalagonist (Beyersdorf et al., 2005). The superagonistic TGN1412 binds to alateral motif of CD28, while the conventional agonistic molecule 9.3binds close to the ligand binding epitope. As a consequence of thedifferent binding epitopes, superagonistic and conventional agonisticantibodies differ in their ability to form linear complexes of CD28molecules on the surface of T cells. Precisely, TGN1412 is able toefficiently form linear arrays of CD28, which presumably leads toaggregated signaling components which are sufficient to surpass thethreshold for T cell activation. The conventional agonist 9.3, on theother hand, leads to complexes which are not linear in structure. Anattempt to convert conventional agonistic binders based on the 9.3 clonehas been previously published (Otz et al., 2009) using a recombinantbi-specific single-chain antibody directed to a melanoma-associatedproteoglycan and CD28. The reported bispecific single chain antibody wasreported to exert “supra-agonistic” activity despite the use of aconventional CD28 agonistic binder 9.3, based in the intrinsic tendencyof bispecific single chain antibodies to form multimeric constructs.

It has been found that a better T cell activation is achieved whenlimiting amounts of anti-CD3 bispecific antibodies, i.e. T cellbispecific antibodies (TCBs) such as CEA-TCB, are combined withagonistic anti-CD28 molecules. Given, that CD28 is expressed at baselineon T cells in various tumor indications (Lavin et al., 2017; Tirosh etal., 2016, Zheng et al., 2017) and activation of CD28 signaling enhancesT cell receptor signals, the combination of a TCB molecule with atumor-targeted CD28 molecule is expected to act synergistically toinduce strong and long-lasting anti-tumor responses. Thus, we hereindescribe novel tumor-targeted agonistic CD28 molecules which displaysynergy with TCBs and require CD28 binding monovalency for strict tumortarget dependence in the presence of TCB signals.

Immunotherapy of Solid Tumors

The treatment of solid tumors is an ongoing challenge, with littleadvancements seen over the last years. Typically, the treatment will bea combination of surgery and chemotherapy and/or radiotherapy. Whilequite a few new treatment modalities have been developed recently, thereis still a need for further improvements, to increase survival rates ofpatients suffering from solid tumors, and improve their quality of life.Solid tumors rarely express one tumor specific antigen. For most solidtumors, it is more common to find a tumor associated antigen (TAA) thatis enriched on tumors but also expressed at very low levels on normaltissues. The TAA is preferably presented on the surface of the solidtumor cell or on a cell of the tumor stroma. This is the case for manyfrequently targeted TAAs for solid tumors, including FibroblastActivation Protein (FAP), Carcinoembryonic Antigen (CEA), Folatereceptor alpha (FolR1), Melanoma-associated Chondroitin SulfateProteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), humanepidermal growth factor receptor 2 (HER2) and p95HER2. Further TAAsinclude HER3, EpCAM, TPBG (5T4), mesothelin, MUC1, and PSMA. Bispecificagonistic CD28 antigen binding molecule comprising an antigen bindingdomain that specifically binds to a tumor associated antigen will thusbe directed primarily to the tumor surface or tumor microenvironment andwill specifically activate T cells in the proximity of the tumor while asystemic activation may be avoided.

Rationale for Targeting CD28 Agonism to B Cell Malignancies

Non-Hodgkin's lymphoma (NHL) is one of the leading causes of cancerdeath in the United States and in Europe. Follicular lymphoma (FL) isindolent in its course and has a slow rate of evolution, with a mediansurvival of 8 to 10 years; patients in advanced clinical stages usuallyare not curable. Likewise, in 2% to 3% of patients per year, thephenotype of FL can transform into an aggressive, large cell lymphoma, acritical event in the course of the disease and one that is associatedwith increased lymphoma-related mortality. Mantle cell lymphoma anddiffuse large B cell lymphoma (DLBCL) are more aggressive and, ifuntreated, have a median survival rate of only 6 months. The lack ofcurative outcomes for many patients with both indolent and aggressiveNHL subtypes remains an unmet medical need, despite significant advancesin immune-therapeutics that have extended progression free-survivaltimes. During the past several years, significant prolonged survival inDLBCL has been observed, particularly with the addition of the anti-CD20monoclonal antibody, rituximab (Rituxan®, MabThera®) to intensivecytotoxic chemotherapeutic regimens. However, despite conventionaltreatment for previously untreated DLBCL is curative in intent, themajority of patients will eventually relapse. Likewise, advanced FLremains largely incurable by current SoC and is characterized byrepeated relapses and progressively shorter remissions. Currently, manynew generation monoclonal antibodies are in different preclinical andclinical phases of assessment to further improve the outcome of NHLpatients and overcome mechanisms of rituximab resistance. High dosechemotherapy with autologous stem cell support or allogeneic stem celltransplantation provides a curative option for only a minority (10%) ofpatients with relapsed/refractory (r/r) DLBCL and is associated withsubstantial treatment-related mortality. Other approaches for thetreatment of NHL currently in development include molecular targetedcompounds like venetoclax and BET-inhibitors. Recently approved novelagents include lenalidomide, idelalisib, and copanlisib. Chimericantigen receptor (CAR) T-cell therapy has been approved for thetreatment of aggressive forms of r/r B-NHL, but this therapy isavailable only in limited settings and can be associated with fatalneurologic events and cytokine release syndrome (CRS). Bispecificantibody constructs redirecting the lysis of cytotoxic cells tomalignant B cells are currently in development and have shown verypromising efficacy against NHL. Chemotherapy-free treatments areenvisioned for the future of NHL and will likely be based on bispecificantibodies or chimeric antigen receptor T cells (CAR T cells). A CD28agonist targeted against a B cell surface antigen in combination withimmunotherapy shall enhance the survival and/or cure rates for patientswith B cell malignancies, without compromising their quality of life.

B Cell Surface Antigens as a Target for B Cell Malignancies

TAAs related to B cell malignancies are B cell surface antigens. Thehuman CD19 antigen is a 95 KDa transmembrane glycoprotein belonging tothe immunoglobulin superfamily. CD19 is classified as a type Itransmembrane protein, with a single transmembrane domain, a cytoplasmicC-terminus, and extracellular N-terminus. In normal cells, it is themost ubiquitously expressed protein in the B lymphocyte lineage. CD19expression is maintained in B-lineage cells that have undergoneneoplastic transformation, and therefore CD19 is useful in diagnosis ofleukemias and lymphomas using monoclonal antibodies (mAbs) and flowcytometry, so does the CD20 antigen. Because B lineage leukemias andlymphomas rarely lose CD19 expression, and because it is not expressedin the pluripotent stem cell, it has become the target for a variety ofimmunotherapeutic agents, including immunotoxins. CD79 is the signalingcomponent of the B-cell receptor consisting of a covalent heterodimercontaining CD79a (Igα, mb-1) and CD79b (Igβ, B29). CD79a and CD79b eachcontain an extracellular immunoglobulin (Ig) domain, a transmembranedomain, and an intracellular signaling domain, an immunoreceptortyrosine-based activation motif (ITAM) domain, like other signallingproteins such as CD3 or activatory Fey receptor. CD79a and CD79b arethus transmembrane proteins that compose the signalling subunits of theB cell receptor (BCR). CD79b is a 39 KDa protein exclusively expressedon B cells and, in cooperation with CD79a, initiates the signaltransduction cascade downstream of the BCR, which leads tointernalization of the BCR complex, its translocation to the endosomes,and antigen presentation. In B cells, antigen-induced BCR clusteringtriggers tyrosine phosphorylation of the ITAM of CD79a and CD79b by Srckinases. This leads to the recruitment and activation of an array ofeffector molecules belonging to the BCR signalling cascade, includingthe most notable SYK and BLNK. Further downstream, recruitment of PLCg2,Btk, and ERK facilitate calcium flux and activate the B cells, which arethen ready to receive additional co-activating signals that will drivetheir proliferation and differentiation into memory or effector cells.During this process, B cells become robust APCs and release cytokinesthat can influence the outcome and quality of the immune response. Inaddition to their role in BCR signalling, the CD79 subunits are alsoessential for the transport and display of membrane-bound Ig from theendoplasmic reticulum to the cell surface. The average surfaceexpression of CD79b on NHLs is similar to that on normal B-cells, butwith a greater range. Given the expression of CD79b, it is beneficial toproduce therapeutic antibodies to the CD79b antigen that create minimalor no antigenicity when administered to patients, especially for chronictreatment.

It has been found that a better T cell activation is achieved whenlimiting amounts of anti-CD3 bispecific antibodies, i.e. T cellbispecific antibodies (TCBs) such as for example a CD20/CD3 bispecificantibody, are combined with agonistic anti-CD28 molecules. Given, thatCD28 is expressed at baseline on T cells in various tumor indications(Lavin et al., 2017; Tirosh et al., 2016, Zheng et al., 2017) andactivation of CD28 signaling enhances T cell receptor signals, thecombination of T cell bispecific antibodies with bispecific agonisticCD28 antigen binding molecules targeting a B cell surface antigen isexpected to act synergistically to induce strong and long-lastinganti-tumor responses. Thus, we herein describe novel bispecificagonistic CD28 antigen binding molecules targeting a B cell surfaceantigen which display synergy with TCBs and require CD28 bindingmonovalency for strict tumor target dependence in the presence of TCBsignals.

Immunotherapy in Multiple Myeloma

Affecting ˜75,000 new patients every year in the EU and US, multiplemyeloma (MM) is one of the most common hematological malignancies withremaining high unmet medical need. Multiple myeloma is characterized byterminally differentiated plasma cells that secrete non-functionalmonoclonal immunoglobulins. In the short-term, the immunomodulatorydrugs such as lenalidomide and pomalidomide, and proteasome inhibitorssuch as carfilzomib or bortezomib may remain the backbone of 1st linetherapy for multiple myeloma (Moreau et al, 2016). However, these drugsdo not target specifically the diseased tumor cells e.g. diseased plasmacells (PC). Efforts have been made towards selectively depleting theplasma cells in multiple myeloma. The lack of surface proteins thatspecifically mark plasma cells has hampered the development ofantibodies or cellular therapies for multiple myeloma. So far, there arefew cases of successful biologics, including daratumumab (anti-CD38) andelotuzumab (anti-CD319), with the caveat that both antigens are alsoexpressed on other normal tissues including hematopoietic lineages andimmune effector cells, which may limit their long-term clinical use. Bcell maturation antigen (BCMA), a transmembrane glycoprotein in thetumor necrosis factor receptor superfamily 17 (TNFRSF17), is expressedat significantly higher levels in all patient MM cells but not on othernormal tissues except normal plasma cells. BCMA-chimeric antigenreceptor (CAR) T-cells have already shown significant clinicalactivities in patients with RRMM who have undergone at least three priortreatments, including a proteasome inhibitor and an immunomodulatoryagent. Additional modalities, including anti-BCMA antibody-drugconjugate also has achieved significant clinical responses in patientswho failed at least three prior lines of therapy, including an anti-CD38antibody, a proteasome inhibitor, and an immunomodulatory agent (Cho etal, 2018). One challenge of e.g. BCMA- or CD38-targeted therapy lies inthe presence of high levels of soluble BCMA or CD38 in the serum of MMpatients, which may reduce the amount of active drug in the patient. Analternative might be new targets, such as the G protein-coupled receptorclass C group 5 member D (GPRC5D), that is differentially expressed byplasma cells in multiple myeloma versus plasma cells from healthydonors, and has no soluble form. It has been reported that GPRC5D isassociated with prognosis and tumor load in multiple myeloma patients(Atamaniuk, J. et al., 2012; and Cohen, Y., et al., 2013). GPRC5D is anorphan receptor with no known ligand(s) and largely unknown biology inmen in general and in cancer specifically. The GPRC5D encoding gene,which is mapped on chromosome12p13.3, contains three exons and spansabout 9.6 kb (Brauner-Osborne, H. et al. 2001). The large first exonencodes the seven-transmembrane domain. It has been shown that GPRC5D isinvolved in keratin formation in hair follicles in animals (Gao, Y. etal., 2016, and Inoue, S. et al., 2004). WO 2018/017786 A2 disclosesGPRC5D-specific antibodies or antigen-binding fragments.

Rationale for Targeting CD28 Agonism to Diseased Plasma Cells inMultiple Myeloma

CD28 agonism in Multiple Myeloma may exert different biologicalfunctions on immune, respective MM plasma cells. While co-activation ofT-cells via CD28 is expected to drive anti-tumor responses, CD28 agonismon MM cells mediates pro-survival signaling via regulation of PI3K/Akt,FoxO3a, and Bimm which in turn is described to induce chemotherapeuticresistance in multiple myeloma (Murray M. E. et al, 2014).Over-expression of CD28 on newly diagnosed Multiple Myeloma plasma cellsis described to correlate with worse clinical outcome (Bahlis et al.,2007). However, while CD28 activation enhances myeloma cell survival,its activation inhibits myeloma cell proliferation. Agonizing CD28 inpresence of a strong immune cell mediated response, such as a T-cellbispecific activation of T-cells, can further boost efficient anti-tumorresponses. We herein provide bispecific agonistic CD28 antigen bindingmolecules that specifically bind a human Multiple Myeloma (MM) cellsurface antigen. Particularly, the bispecific agonistic CD28 antigenbinding molecules according to the invention targeting the TAAs selectedfrom BCMA, CD38 and GPRC5D and CD28 expressed on T-cells have thepotency to treat multiple myeloma as single agent or in combination withother agents such as T cell bispecific antibodies (TCBs) targeting ahuman MM cell surface antigen.

SUMMARY

The present invention describes tumor-targeted bispecific agonistic CD28antigen binding molecules which achieve a tumor-dependent T cellactivation and tumor cell killing without the necessity to formmultimers. The bispecific CD28 antigen binding molecules of the presentinvention are characterized by monovalent binding to CD28 and in thatthey comprise at least one antigen binding domain capable of specificbinding to a tumor-associated antigen (such as Fibroblast ActivationProtein (FAP) or Carcinoembryonic Antigen (CEA), CD19 or GPRC5D).Furthermore, they possess an Fc domain composed of a first and a secondsubunit capable of stable association comprising one or more amino acidsubstitution that reduces the binding affinity of the antigen bindingmolecule to an Fc receptor and/or effector function. Fcreceptor-mediated cross-linking is thereby abrogated and tumor-specificactivation is achieved by cross-linking through binding of the at leastone antigen binding domain capable of specific binding to atumor-associated antigen to its antigen.

Thus, the invention provides a bispecific agonistic CD28 antigen bindingmolecule characterized by monovalent binding to CD28, comprising

-   -   (a) one antigen binding domain capable of specific binding to        CD28,    -   (b) at least one antigen binding domain capable of specific        binding to a tumor-associated antigen, and    -   (c) an Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, a bispecific agonistic CD28 antigen binding molecule asdefined below is provided, wherein the Fc domain is an IgG, particularlyan IgG1 Fc domain or an IgG4 Fc domain. In one particular aspect, the Fcdomain composed of a first and a second subunit capable of stableassociation is an IgG1 Fc domain. In one aspect, the Fc domain comprisesthe amino acid substitutions L234A and L235A (numbering according toKabat EU index). In one aspect, the Fc domain is of human IgG1 subclassand comprises the amino acid mutations L234A, L235A and P329G (numberingaccording to Kabat EU index).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as defined herein before, wherein the antigen binding domaincapable of specific binding to CD28 comprises

-   -   (i) a heavy chain variable region (V_(H)CD28) comprising a heavy        chain complementary determining region CDR-H1 of SEQ ID NO: 36,        a CDR-H2 of SEQ ID NO: 37, and a CDR-H3 of SEQ ID NO: 38, and a        light chain variable region (V_(L)CD28) comprising a light chain        complementary determining region CDR-L1 of SEQ ID NO: 39, a        CDR-L2 of SEQ ID NO: 40 and a CDR-L3 of SEQ ID NO: 41; or    -   (ii) a heavy chain variable region (V_(H)CD28) comprising a        CDR-H1 of SEQ ID NO: 20, a CDR-H2 of SEQ ID NO: 21, and a CDR-H3        of SEQ ID NO: 22, and a light chain variable region (V_(L)CD28)        comprising a CDR-L1 of SEQ ID NO: 23, a CDR-L2 of SEQ ID NO: 24        and a CDR-L3 of SEQ ID NO: 25.

In one aspect, the antigen binding domain capable of specific binding toCD28 of the bispecific agonistic CD28 antigen binding molecule comprisesa heavy chain variable region (V_(H)CD28) comprising a CDR-H1 of SEQ IDNO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3 of SEQ ID NO: 38, and alight chain variable region (V_(L)CD28) comprising a CDR-L1 of SEQ IDNO: 39, a CDR-L2 of SEQ ID NO: 40 and a CDR-L3 of SEQ ID NO: 41.

In another aspect, the antigen binding domains capable of specificbinding to CD28 of the bispecific agonistic CD28 antigen bindingmolecule comprises a heavy chain variable region (V_(H)CD28) comprisinga CDR-H1 of SEQ ID NO: 20, a CDR-H2 of SEQ ID NO: 21, and a CDR-H3 ofSEQ ID NO: 22, and a light chain variable region (V_(L)CD28) comprisinga CDR-L1 of SEQ ID NO: 23, a CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQID NO: 25.

Furthermore, provided is a bispecific agonistic CD28 antigen bindingmolecule as defined herein before, wherein the antigen binding domaincapable of specific binding to CD28 comprises a heavy chain variableregion (V_(H)CD28) comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:26, and a light chain variable region (V_(L)CD28)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:27.

In a further aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 andSEQ ID NO:51, and a light chain variable region (V_(L)CD28) comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:27, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ IDNO:61.

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule, wherein the antigen binding domain capable of specificbinding to CD28 comprises

-   -   (a) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (b) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (c) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:51 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:61, or    -   (d) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (e) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (f) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (g) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (h) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:43 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (j) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (k) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27.

In one particular aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:47 and alight chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:54.

In another particular aspect, a bispecific agonistic CD28 antigenbinding molecule is provided, wherein the antigen binding domain capableof specific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:46 and alight chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:53.

In a further particular aspect, a bispecific agonistic CD28 antigenbinding molecule is provided, wherein the antigen binding domain capableof specific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:42 and alight chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:27.

In one aspect, a bispecific agonistic CD28 antigen binding molecule isprovided, wherein the antigen binding domain capable of specific bindingto a tumor-associated antigen is an antigen binding domain capable ofspecific binding to Carcinoembryonic Antigen (CEA).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to CEA comprises

-   -   (i) a heavy chain variable region (V_(H)CEA) comprising a CDR-H1        comprising the amino acid sequence of SEQ ID NO:188, a CDR-H2        comprising the amino acid sequence of SEQ ID NO:189, and a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:190, and        a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:191, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:192, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:193; or    -   (ii) a heavy chain variable region (V_(H)CEA) comprising a        CDR-H1 comprising the amino acid sequence of SEQ ID NO:180, a        CDR-H2 comprising the amino acid sequence of SEQ ID NO:181, and        a CDR-H3 comprising the amino acid sequence of SEQ ID NO:182,        and a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:183, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:184, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:185; or    -   (iii) a heavy chain variable region (V_(H)CEA) comprising a        CDR-H1 comprising the amino acid sequence of SEQ ID NO:127, a        CDR-H2 comprising the amino acid sequence of SEQ ID NO:128, and        a CDR-H3 comprising the amino acid sequence of SEQ ID NO:129,        and a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:130, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:131, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:132, or    -   (iv) a heavy chain variable region (V_(H)CEA) comprising a        CDR-H1 comprising the amino acid sequence of SEQ ID NO:507, a        CDR-H2 comprising the amino acid sequence of SEQ ID NO:508, and        a CDR-H3 comprising the amino acid sequence of SEQ ID NO:509,        and a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:510, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:511, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:512.

In one aspect, the antigen binding domain capable of specific binding toCEA comprises a heavy chain variable region (V_(H)CEA) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:133, and a lightchain variable region (V_(L)CEA) comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:134. Particularly, the antigen binding domaincapable of specific binding to CEA comprises a heavy chain variableregion (V_(H)CEA) comprising the amino acid sequence of SEQ ID NO:186,and a light chain variable region (V_(L)CEA) comprising the amino acidsequence of SEQ ID NO:187.

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule, wherein the antigen binding domain capable of specificbinding to CEA comprises

-   -   (a) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:194 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:195, or    -   (b) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:196 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:197, or    -   (c) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:198 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:199, or    -   (d) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:200 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:201, or    -   (e) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:202 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:203, or    -   (f) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:204 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:205, or    -   (g) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:206 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:207, or    -   (h) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:208 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:209, or    -   (i) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:210 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:211, or    -   (j) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:212 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:213.

Particularly, the antigen binding domain capable of specific binding toCEA comprises a heavy chain variable region (V_(H)CEA) comprising theamino acid sequence of SEQ ID NO:200 and a light chain variable region(V_(L)CEA) comprising the amino acid sequence of SEQ ID NO:201.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to Fibroblast Activation Protein (FAP). Inone aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to FAP comprises (a) a heavy chain variable region(V_(H)FAP) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:12, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:13, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:14, and a light chain variable region (V_(L)FAP) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:15, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:16, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:17, or (b) a heavy chainvariable region (V_(H)FAP) comprising (i) CDR-H1 comprising the aminoacid sequence of SEQ ID NO:4, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:5, and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO:6, and a light chain variable region (V_(L)FAP)comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:7, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9. Inparticular, the antigen binding domain capable of specific binding toFAP comprises a heavy chain variable region (V_(H)FAP) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:13, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:14, and a light chainvariable region (V_(L)FAP) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:15, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:16, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:17. In one aspect, a bispecific agonistic CD28antigen binding molecule is provided, wherein the antigen binding domaincapable of specific binding to FAP comprises (a) a heavy chain variableregion (V_(H)FAP) comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:18, and a light chain variable region (V_(L)FAP)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:19,or (b) a heavy chain variable region (V_(H)FAP) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:10, and a light chainvariable region (V_(L)FAP) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:11. Particularly, the antigen binding domaincapable of specific binding to FAP comprises a heavy chain variableregion (V_(H)FAP) comprising the amino acid sequence of SEQ ID NO:18 anda light chain variable region (V_(L)FAP) comprising the amino acidsequence of SEQ ID NO:19.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to epithelial cell adhesion molecule(EpCAM). In one aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, wherein the antigen binding domaincapable of specific binding to EpCAM comprises a heavy chain variableregion (V_(H)EpCAM) comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:515, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:516, and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO:517, and a light chain variable region(V_(L)EpCAM) comprising (iv) CDR-L1 comprising the amino acid sequenceof SEQ ID NO:518, (v) CDR-L2 comprising the amino acid sequence of SEQID NO:519, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:520. In one aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to EpCAM comprises (a) a heavy chain variable region(V_(H)EpCAM) comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:521, and a light chain variable region (V_(L)EpCAM) comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:522.Particularly, the antigen binding domain capable of specific binding toEpCAM comprises a heavy chain variable region (V_(H)EpCAM) comprisingthe amino acid sequence of SEQ ID NO:521 and a light chain variableregion (V_(L)EpCAM) comprising the amino acid sequence of SEQ ID NO:522.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to HER3. In one aspect, provided is abispecific agonistic CD28 antigen binding molecule as described herein,wherein the antigen binding domain capable of specific binding to HER3comprises a heavy chain variable region (V_(H)HER3) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:523, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:524, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:525, and a light chainvariable region (V_(L)HER3) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:526, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:527, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:528. In one aspect, a bispecific agonistic CD28antigen binding molecule is provided, wherein the antigen binding domaincapable of specific binding to HER3 comprises (a) a heavy chain variableregion (V_(H)HER3) comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:529, and a light chain variable region (V_(L)HER3)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:530.Particularly, the antigen binding domain capable of specific binding toHER3 comprises a heavy chain variable region (V_(H)HER3) comprising theamino acid sequence of SEQ ID NO:529 and a light chain variable region(V_(L)HER3) comprising the amino acid sequence of SEQ ID NO:530.

In yet another aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to a tumor-associated antigen is an antigen bindingdomain capable of specific binding to CD30. In one aspect, provided is abispecific agonistic CD28 antigen binding molecule as described herein,wherein the antigen binding domain capable of specific binding to CD30comprises a heavy chain variable region (V_(H)CD30) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:531, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:532, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:533, and a light chainvariable region (V_(L)CD30) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:534, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:535, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:536. In one aspect, a bispecific agonistic CD28antigen binding molecule is provided, wherein the antigen binding domaincapable of specific binding to CD30 comprises (a) a heavy chain variableregion (V_(H)CD30) comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:537, and a light chain variable region (V_(L)CD30)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:538.Particularly, the antigen binding domain capable of specific binding toCD30 comprises a heavy chain variable region (V_(H)CD30) comprising theamino acid sequence of SEQ ID NO:537 and a light chain variable region(V_(L)CD30) comprising the amino acid sequence of SEQ ID NO:538.

Furthermore, provided is a bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto a tumor-associated antigen is an antigen binding domain capable ofspecific binding to TBPG. In one aspect, provided is a bispecificagonistic CD28 antigen binding molecule as described herein, wherein theantigen binding domain capable of specific binding to TBPG comprises aheavy chain variable region (V_(H)TBPG) comprising (i) CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:539, (ii) CDR-H2 comprising theamino acid sequence of SEQ ID NO:540, and (iii) CDR-H3 comprising theamino acid sequence of SEQ ID NO:541, and a light chain variable region(V_(L)TBPG) comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:542, (v) CDR-L2 comprising the amino acid sequence of SEQ IDNO:543, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:544. In one aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to TBPG comprises (a) a heavy chain variable region(V_(H)TBPG) comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:545, and a light chain variable region (V_(L)TBPG) comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:546.Particularly, the antigen binding domain capable of specific binding toTBPG comprises heavy chain variable region (V_(H)TBPG) comprising theamino acid sequence of SEQ ID NO:545 and a light chain variable region(V_(L)TBPG) comprising the amino acid sequence of SEQ ID NO:546.

In a further aspect, the invention provides a bispecific agonistic CD28antigen binding molecule characterized by monovalent binding to CD28,comprising (a) one antigen binding domain capable of specific binding toCD28, (b) at least one antigen binding domain capable of specificbinding to a Multiple Myeloma (MM) cell surface antigen, and (c) an Fcdomain composed of a first and a second subunit capable of stableassociation comprising one or more amino acid substitution that reducesthe binding affinity of the antigen binding molecule to an Fc receptorand/or effector function. In one aspect, the Multiple Myeloma (MM) cellsurface antigen selected from the group consisting of CD38, BCMA andGPRC5D.

Thus, provided is a bispecific agonistic CD28 antigen binding molecule,wherein the antigen binding domain capable of specific binding to atumor-associated antigen is an antigen binding domain capable ofspecific binding to GPRC5D. In one aspect, provided is a bispecificagonistic CD28 antigen binding molecule as described herein, wherein theantigen binding domain capable of specific binding to GPRC5D comprises(a) a heavy chain variable region (V_(H)GPRC5D) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:563, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:564, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:565, and a light chainvariable region (V_(L)GPRC5D) comprising (iv) CDR-L1 comprising theamino acid sequence of SEQ ID NO:566, (v) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:567, and (vi) CDR-L3 comprising the aminoacid sequence of SEQ ID NO:568, or (b) a heavy chain variable region(V_(H)GPRC5D) comprising (i) CDR-H1 comprising the amino acid sequenceof SEQ ID NO:579, (ii) CDR-H2 comprising the amino acid sequence of SEQID NO:580, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:581, and a light chain variable region (V_(L)GPRC5D) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:582, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:583, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:584. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to GPRC5Dcomprises a heavy chain variable region (V_(H)GPRC5D) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:569, and a lightchain variable region (V L GPRC5D) comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO:570. Particularly, the antigen bindingdomain capable of specific binding to GPRC5D comprises a heavy chainvariable region (V_(H)GPRC5D) comprising the amino acid sequence of SEQID NO:569 and a light chain variable region (V_(L)GPRC5D) comprising theamino acid sequence of SEQ ID NO:570.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto a tumor-associated antigen is an antigen binding domain capable ofspecific binding to CD38. In one aspect, provided is a bispecificagonistic CD28 antigen binding molecule as described herein, wherein theantigen binding domain capable of specific binding to CD38 comprises aheavy chain variable region (V_(H)CD38) comprising (i) CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:547, (ii) CDR-H2 comprising theamino acid sequence of SEQ ID NO:548, and (iii) CDR-H3 comprising theamino acid sequence of SEQ ID NO:549, and a light chain variable region(V_(L)CD38) comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:550, (v) CDR-L2 comprising the amino acid sequence of SEQ IDNO:551, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:552. In one aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to CD38 comprises a heavy chain variable region(V_(H)CD38) comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:553, and a light chain variable region (V_(L)CD38) comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:554.Particularly, the antigen binding domain capable of specific binding toCD38 comprises a heavy chain variable region (V_(H)CD38) comprising theamino acid sequence of SEQ ID NO:553 and a light chain variable region(V_(L)CD38) comprising the amino acid sequence of SEQ ID NO:554.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto a tumor-associated antigen is an antigen binding domain capable ofspecific binding to BCMA. In one aspect, provided is a bispecificagonistic CD28 antigen binding molecule as described herein, wherein theantigen binding domain capable of specific binding to BCMA comprises aheavy chain variable region (V_(H)BCMA) comprising (i) CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:555, (ii) CDR-H2 comprising theamino acid sequence of SEQ ID NO:556, and (iii) CDR-H3 comprising theamino acid sequence of SEQ ID NO:557, and a light chain variable region(V L BCMA) comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:558, (v) CDR-L2 comprising the amino acid sequence of SEQ IDNO:559, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:560. In one aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to BCMA comprises a heavy chain variable region(V_(H)BCMA) comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:559, and a light chain variable region (V_(L)BCMA) comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:560.Particularly, the antigen binding domain capable of specific binding toBCMA comprises a heavy chain variable region (V_(H)BCMA) comprising theamino acid sequence of SEQ ID NO:561 and a light chain variable region(V_(L)BCMA) comprising the amino acid sequence of SEQ ID NO:562.

In a further aspect, the invention provides a bispecific agonistic CD28antigen binding molecule characterized by monovalent binding to CD28,comprising (a) one antigen binding domain capable of specific binding toCD28, (b) at least one antigen binding domain capable of specificbinding to a B cell surface antigen, and (c) an Fc domain composed of afirst and a second subunit capable of stable association comprising oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or effector function. Inone aspect, the B cell surface antigen selected from the groupconsisting of CD19, CD79b, CD20, CD22 and CD37.

Thus, provided is a bispecific agonistic CD28 antigen binding molecule,wherein the antigen binding domain capable of specific binding to atumor-associated antigen is an antigen binding domain capable ofspecific binding to CD19. In one aspect, provided is a bispecificagonistic CD28 antigen binding molecule as described herein, wherein theantigen binding domain capable of specific binding to CD19 comprises (a)a heavy chain variable region (V_(H)CD19) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:406, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:407, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:408, and a light chainvariable region (V_(L)CD19) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:409, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:410, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:411, or (b) a heavy chain variable region(V_(H)CD19) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:414, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:415, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:416, and a light chain variable region (V_(L)CD19) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:417, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:418, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:419. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to CD19 comprises(a) a heavy chain variable region (V_(H)CD19) comprising an amino acidsequence that is at least about 95%, 98% or 100% identical to the aminoacid sequence of SEQ ID NO:412, and a light chain variable region(V_(L)CD19) comprising an amino acid sequence that is at least about95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:413,or (b) a heavy chain variable region (V_(H)CD19) comprising an aminoacid sequence that is at least about 95%, 98% or 100% identical to theamino acid sequence of SEQ ID NO:420, and a light chain variable region(V_(L)CD19) comprising an amino acid sequence that is at least about95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:421.Particularly, the antigen binding domain capable of specific binding toCD19 comprises a heavy chain variable region (V_(H)CD19) comprising anamino acid sequence of SEQ ID NO:412 and a light chain variable region(V_(L)CD19) comprising an amino acid sequence of SEQ ID NO:413.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto a tumor-associated antigen is an antigen binding domain capable ofspecific binding to CD79b. In one aspect, provided is a bispecificagonistic CD28 antigen binding molecule as described herein, wherein theantigen binding domain capable of specific binding to CD79b comprises aheavy chain variable region (V_(H)CD79b) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:422, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:423, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:424, and a light chainvariable region (V_(L)CD79b) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:425, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:426, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:427. In one aspect, a bispecific agonistic CD28antigen binding molecule is provided, wherein the antigen binding domaincapable of specific binding to CD79b comprises a heavy chain variableregion (V_(H)CD79b) comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:428, and a light chain variable region(V_(L)CD79b) comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:429. Particularly, the antigen binding domain capable ofspecific binding to CD79b comprises a heavy chain variable region(V_(H)CD79b) comprising the amino acid sequence of SEQ ID NO:428, and alight chain variable region (V_(L)CD79b) comprising the amino acidsequence of SEQ ID NO:429.

In a further aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as defined herein before, wherein the antigen bindingdomain capable of specific binding to CD28 is a Fab fragment or acrossFab fragment.

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In a further aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second Fab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of one of        the Fc domain subunits.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeas disclosed herein is provided, comprising

-   -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second and a third Fab fragment capable of specific        binding to a tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of the        first Fc domain subunit, and the third Fab fragment capable of        specific binding to a tumor-associated antigen is fused at the        C-terminus of the Fab heavy chain to the N-terminus of the        second Fc domain subunit.

In a further aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) a Fab fragment capable of specific binding to CD28,    -   (b) a VH and VL domain capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,        wherein the Fab fragment capable of specific binding to CD28 is        fused at its C-terminus to the N-terminus of the first Fc domain        subunit, and wherein one of the VH and VL domain capable of        specific binding to a tumor-associated antigen is fused via a        peptide linker to the C-terminus of the first Fc domain subunit        and the other one of the VH and VL domain capable of specific        binding to a tumor-associated antigen is fused via a peptide        linker to the C-terminus of the second Fc domain subunit.

According to another aspect of the invention, there is provided one ormore isolated polynucleotide(s) encoding the bispecific agonistic CD28antigen binding molecule of the invention. The invention furtherprovides one or more vector(s), particularly expression vector(s),comprising the isolated polynucleotide(s) of the invention, and a hostcell comprising the isolated polynucleotide(s) or the expressionvector(s) of the invention. In some aspects, the host cell is aeukaryotic cell, particularly a mammalian cell. In another aspect,provided is a method of producing a bispecific agonistic CD28 antigenbinding molecule as described herein comprising culturing the host cellof the invention under conditions suitable for the expression of thebispecific agonistic CD28 antigen binding molecule. Optionally, themethod also comprises recovering the bispecific agonistic CD28 antigenbinding molecule. The invention also encompasses a bispecific agonisticCD28 antigen binding molecule produced by the method of the invention.

The invention further provides a pharmaceutical composition comprising abispecific agonistic CD28 antigen binding molecule of the invention andat least one pharmaceutically acceptable excipient. In one aspect, thepharmaceutical composition is for use in the treatment of cancer.

Also encompassed by the invention are methods of using the bispecificagonistic CD28 antigen binding molecule and the pharmaceuticalcomposition of the invention. In one aspect the invention provides abispecific agonistic CD28 antigen binding molecule or a pharmaceuticalcomposition according to the invention for use as a medicament. In oneaspect, provided is a bispecific agonistic CD28 antigen binding moleculeas described herein for use in (a) enhancing cell activation or (b)enhancing T cell effector functions. In one aspect, provided is abispecific agonistic CD28 antigen binding molecule or a pharmaceuticalcomposition according to the invention for use in the treatment of adisease. In a specific aspect, the disease is cancer. In another aspectis provided a bispecific agonistic CD28 antigen binding molecule orpharmaceutical composition according to the invention is for use in thetreatment of cancer, wherein the bispecific agonistic CD28 antigenbinding molecule is administered in combination with a chemotherapeuticagent, radiation therapy and/or other agents for use in cancerimmunotherapy. In a further aspect, provided is a bispecific agonisticCD28 antigen binding molecule or a pharmaceutical composition for use inthe treatment of cancer, wherein the bispecific agonistic CD28 antigenbinding molecule is administered in combination with a T-cell activatinganti-CD3 bispecific antibody. In yet another aspect, provided is abispecific agonistic CD28 antigen binding molecule or a pharmaceuticalcomposition for use in the treatment of cancer, wherein the bispecificagonistic CD28 antigen binding molecule is administered in combinationwith an anti-PD-L1 antibody or an anti-PD-1 antibody.

Also provided is the use of a bispecific agonistic CD28 antigen bindingmolecule according to the invention in the manufacture of a medicamentfor the treatment of a disease; as well as a method of treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a bispecific agonistic CD28 antigenbinding molecule according to the invention or a composition comprisingthe bispecific agonistic CD28 antigen binding molecule according to theinvention in a pharmaceutically acceptable form. In a specific aspect,the disease is cancer. In one aspect, provided is a method (a) enhancingcell activation or (b) enhancing T cell effector functions in anindividual, comprising administering a bispecific agonistic CD28 antigenbinding molecule according to the invention or a composition comprisingthe bispecific agonistic CD28 antigen binding molecule according to theinvention in a pharmaceutically acceptable form to said individual. Inanother aspect, provided is the use of a bispecific agonistic CD28antigen binding molecule according to the invention in the manufactureof a medicament for the treatment of a disease, wherein the treatmentcomprises co-administration with a chemotherapeutic agent, radiationtherapy and/or other agents for use in cancer immunotherapy. In afurther aspect, provided is a method of treating a disease in anindividual, comprising administering to said individual atherapeutically effective amount of a bispecific agonistic CD28 antigenbinding molecule according to the invention or a composition comprisingthe bispecific agonistic CD28 antigen binding molecule according to theinvention in a pharmaceutically acceptable form, wherein the methodcomprises co-administration with a chemotherapeutic agent, radiationtherapy and/or other agents for use in cancer immunotherapy. In afurther aspect, provided is a method of treating a disease in anindividual, comprising administering to said individual atherapeutically effective amount of a bispecific agonistic CD28 antigenbinding molecule according to the invention or a composition comprisingthe bispecific agonistic CD28 antigen binding molecule according to theinvention in a pharmaceutically acceptable form, wherein the methodcomprises co-administration of a T-cell activating anti-CD3 bispecificantibody. In another aspect, provided is a method of treating a diseasein an individual, comprising administering to said individual atherapeutically effective amount of a bispecific agonistic CD28 antigenbinding molecule according to the invention or a composition comprisingthe bispecific agonistic CD28 antigen binding molecule according to theinvention in a pharmaceutically acceptable form, wherein the methodcomprises co-administration of an anti-PD-L1 antibody or an anti-PD-1antibody. Also provided is a method of inhibiting the growth of tumorcells in an individual comprising administering to the individual aneffective amount of the bispecific agonistic CD28 antigen bindingmolecule according to the invention, or a composition comprising thebispecific agonistic CD28 antigen binding molecule according to theinvention in a pharmaceutically acceptable form, to inhibit the growthof the tumor cells. In any of the above aspects, the individualpreferably is a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIGS. 1A to 1L schematic illustrations of the molecules as describedare shown.

FIG. 1A shows the CD28 agonistic antibody CD28(SA) in its huIgG4 isoform(TGN1412).

FIG. 1B illustrates the CD28(SA) agonistic antibody as hu IgG1 PGLALAisotype (“Fc silent”).

Bispecific FAP-CD28 antigen binding molecules in 1+1 format, 1+2 format,2+2 format and 1+4 format are shown in FIGS. 1C, 1D, 1E and IF,respectively.

Bispecific CEA-CD28 antigen binding molecules in 1+2 format, 2+2 formatand 1+1 format are shown in FIGS. 1G, 1H and 1J, respectively.

FIG. 1I shows a schematic illustration of the CD28 agonistic antibodyvariants as monovalent hu IgG1 PGLALA isotype (“Fc silent”).

FIG. 1K shows a bispecific FAP-CD28 antigen binding molecule in 1+1format, wherein the FAP antigen binding domain is represented as VH andVL domains each fused to one C-terminus of the Fc domain subunits.

FIG. 1L illustrates a bispecific FAP-CD28 antigen binding molecule in2+1 format, wherein the CD28 antigen binding domain is represented ascrossFab that is fused at its C-terminus to the N-terminus of one of theheavy chains of the “bivalent” FAP antibody.

FIG. 1M shows another bispecific FAP-CD28 antigen binding molecule in1+1 format, wherein the CD28 antigen binding domain is represented ascrossFab that is fused at its C-terminus to the N-terminus of the Fabfragment binding to FAP.

FIG. 1N illustrates a trispecific FAP-CEA-CD28 antigen binding moleculein 1+1+1 format, wherein the CD28 antigen binding domain is representedas Fab that is fused at the C-terminus of both the light and the heavychain to the N-terminus of both the light and heavy chain of theanti-FAP antigen binding domain on the huIgG1 PG-LALA Fc knob chain andwherein the anti-CEA CrossFab fragment is part of the huIgG1 PG-LALA Fchole chain.

FIGS. 2A, 2B, 2C, 2D and 2E relate to the binding of CD28 agonisticantibodies and FAP-CD28 antigen binding molecules to human CD28 or humanFAP on cells. Shown is the binding of CD28(SA) in it IgG4 isoform vs. huIgG1 PGLALA isotype ti human CD28 in FIG. 2A and the binding ofdifferent FAP-CD28 molecules to human CD28 (FIG. 2B) and human FAP (FIG.2C) on cells. Median fluorescence intensities of binding of differentCD28 agonistic antibodies or anti-DP47 targeted molecules to CHO cellsexpressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61, modifiedto stably overexpress human CD28) or 3T3 cells expressing human FAP(NIH/3T3 cell line (ATCC CRL-1658)) was assessed by flow cytometry.Depicted are technical triplicates with SEM. A comparison ofFAP(4B9)-CD28(SA) antigen binding molecules (Molecules D, E and F asdescribed in Example 1) is shown in FIG. 2D (binding to human CD28) andFIG. 2E (binding to human FAP).

FIGS. 2F and 2G show the binding of different formats of FAP-CD28antigen binding molecules with monovalent binding to CD28 and FAP,respectively. Shown are the curves for FAP-CD28 CTF 1+1 (P1AE2236,Molecule I), FAP-CD28 1+1 (P1AD4492, Molecule C), FAP-CD28 H2T 1+1(P1AE2021, Molecule H), and the two compounds FAP-CD28(SA) 1+2(P1AD9011, Molecule E) and DP47 as reference. Shown are medianfluorescence intensities of binding of the FAP-CD28 antibodies oranti-DP47 antibody (negative control) to CHO cells expressing human CD28(parental cell line CHO-k1 ATCC #CCL-61, modified to stably overexpresshuman CD28) (FIG. 2F) or 3T3 cells expressing human FAP (NIH/3T3 cellline (ATCC CRL-1658)) (FIG. 2G), assessed by flow cytometry. Shown aretechnical triplicates with SEM. FIGS. 2H and 2I show the binding ofFAP-CD28 2+1 (P1AE5231, Molecule G) to CD28 and FAP, respectively.

The alignment of the variable domains of CD28(SA) and variants thereofis shown in FIGS. 3A to 3D. Alignment of the CD28(SA) VH domain andvariants thereof in order to remove cysteine 50 and to reduce theaffinity of the resulting anti-CD28 binders to different degrees isshown in FIGS. 3A and 3B. Of note, in VH variants i and j, the CDRs ofCD28(SA) were grafted from an IGHV1-2 framework into an IGHV3-23framework (FIG. 3B). In FIGS. 3C and 3D, alignment of the CD28(SA) VLdomain and variants thereof in order to reduce the affinity of theresulting anti-CD28 binders to different degrees is shown. In variant t,the CDRs were grafted into the framework sequence of the trastuzumab(Herceptin) VL sequence.

In FIGS. 4A to 4C the binding of affinity-reduced CD28 agonisticantibody variants in monospecific, monovalent IgG formats fromsupernatants to human CD28 on cells is shown. Median fluorescenceintensities of binding to CHO cells expressing human CD28 (parental cellline CHO-k1 ATCC #CCL-61, modified to stably overexpress human CD28)compared to the negative control (anti-DP47) and the original TGN1412,were assessed by flow cytometry. The binding curves of variants 1-10 areshown in FIG. 4A, those of variants 11 to 22 in FIG. 4B and those ofvariants 23 to 31 in FIG. 4C. Depicted are technical duplicates with SD.

In FIGS. 4D and 4E, the binding of FAP-targeted bispecific CD28agonistic antibody variants in huIgG1 PG-LALA 1+1 format with selectedaffinity-reduced CD28 agonistic antibody variants to human CD28 on cellsis shown. The binding curves of bispecific 1+1 constructs with variants8, 11, 12, 15, 16 and 17 are shown in FIG. 4D, whereas the bindingcurves of bispecific 1+1 constructs with variants 19, 23, 25, 27 and 29are shown in FIG. 4E. Selected binders were chosen based on affinitiesfor production in a 1+1, bispecific FAP-targeted format. In FIGS. 4F and4G, the binding of the same FAP-targeted bispecific CD28 agonisticantibody variants in huIgG1 PG-LALA 1+1 format to human FAP is shown.Provided are median fluorescence intensities of binding to CHO cellsexpressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61, modifiedto stably overexpress human CD28) or to 3T3 cells expressing human FAP(NIH/3T3 cell line (ATCC CRL-1658)) compared to the negative control(anti-DP47) and to TGN1412 (Molecule A), assessed by flow cytometry.Shown are technical triplicates with SEM.

The in vitro potency of selected FAP-targeted bispecific CD28 agonisticantibody variants in huIgG1 PG-LALA 1+1 format is illustrated in FIGS.4H, 4I and 4J. PBMC T cells were incubated with MCSP- and FAP-expressingMV3 melanoma cells for 5 days in the presence of limiting concentrationof MCSP-TCB (5 pM, P1AD2189) and increasing concentration of FAP-CD28constructs with the indicated CD28 variant binders. In FIG. 4H is shownthe CFSE-dilution as measure for T cell proliferation of CD8 T cells,assessed by flow cytometry. Error bars show SEM, graphs depict technicaltriplicates of representative results from 2 donors. In FIG. 4I is shownthe correlation of K_(D) (nM) of the CD28 binder variant in relation topotency by area under the curve of (a) as % of the parental TGN1412clone (CD28(SA)). In FIG. 4J the target cell killing at 90 h is shown.

FIGS. 5A to 5D refer to the establishment of high-density (HD)pre-culture and mode of action of CD28(SA). PBMC T cells were eitherpre-cultured at high density (HD) for 2 days or used fresh from PBMCisolation and stimulated with increasing concentrations of CD28(SA).Depicted is CFSE-dilution as proxy for T cell proliferation after 5 daysof stimulation with CD28(SA) (Molecule A, P1AE1975) (FIG. 5A) andcytokine secretion after 2 days (Figure of stimulation. FIG. 5C showsthe percentage of FcγRIIb expression in PBMC monocytes and B cellsbefore and after 2 days HD PBMC pre-culture, assessed by flow cytometry.FIG. 5D: HD pre-cultured PBMCs were co-cultured with CD28(SA) for 5 daysin presence or absence of an FcγRIIb blocking antibody or isotypecontrol and percentage of CF SE-dilution of CD4 T cells was assessed byflow cytometry. Graphs are representative of at least 6 donors (FIG. 5A,and 2 donors (FIG. 5C, 5D), each assessed in independent experiments.The graphs show technical triplicates. Error bars indicate SEM.Statistical analysis was performed by student's t-test. ***: p<0.001.Superagonism of CD28(SA) IgG4 depends on cross-linking to FcγRIIb.

In FIGS. 6A and 6B the T cell proliferation, i.e. CFSE-dilution of CD4 Tcells after 5 days of stimulation with either original Fc wild-type IgG4CD28(SA) (P1AE1975) or CD28(SA) bearing the P329G-LALA mutation(P1AD9289) is shown. T cells were pre-cultured at high density for 2days. Graphs are representative of at least 3 independent experiments.Technical triplicates are shown. Fc-silencing abolishes superagonism inTGN1412. Adding a tumor-targeting moiety to Fc-silenced TGN1412 restoressuperagonism, which is then dependent on the presence of thetumor-target.

In FIGS. 7A, 7B, 7C and 7D a comparison of FAP-targeted CD28 agonists indifferent formats (2+2 and 1+2) and with superagonistic (CD28(SA))binders and conventional agonistic binders (9.3, CD28(CA)) is shown.FAP-targeted CD28 agonists with conventional CD28 agonistic binders donot function as superagonists. PBMC T cells were co-cultured with3T3-huFAP cells (FAP present) in the presence of increasingconcentrations of the FAP-CD28 formats with superagonistic binders (SA,FIG. 7A) or conventional agonistic binders (9.3, FIG. 7B) for 5 days. Tcell proliferation is shown. PBMC T cells were then also co-culturedwith 3T3 WT cells (FAP absent), in the presence of increasingconcentrations of the FAP-CD28 formats with superagonistic binders (SA,FIG. 7C) or conventional agonistic binders (9.3, FIG. 7D) for 5 days.Depicted is CFSE-dilution as measure for T cell proliferation of CD8 Tcells, assessed by flow cytometry on day 5 post stimulation. Graphs showcumulative data from 3 donors in 3 independent experiments. Error barsshow SEM. In the same experimental setup also cytokines were measuredfrom supernatants after 2 days of co-culture. The values are provided inFIG. 7E.

The ability of FAP-CD28 in various formats with either superagonisticCD28(SA) binders or conventional agonistic binders (CD28(CA)) to inducekilling of FAP-expressing RFP-MV3 melanoma cells was assessed over thecourse of 90 h by live cell imaging using the IncuCyte technology. Allmolecules including the FAP-TCB (P1AD4645) were used at 10 nM. FIGS. 8A,8B and 8C show representative results from three donors with technicaltriplicates, respectively. FIG. 8D shows the cumulative resultsexpressed as area under the curve (AUC) at t=90 h of 3 donors from 3independent experiments. Boxes display 25th-75th percentiles, whiskersdisplay min to max. Statistical analysis was performed by paired 1-wayANOVA. ***: p<0.001, ns: not significant.

A comparison of CEA-targeted CD28 agonists in different formats withsuperagonistic and conventional agonistic binders is shown in FIGS. 9Aand 9B. The ability of CEA-CD28 in various formats with eithersuperagonistic CD28(SA) binders or conventional agonistic binders(CD28(CA)) to induce killing of CEA-expressing RFP⁺ MKN45 gastric cancercells was assessed over the course of 90 h by live cell imaging usingthe IncuCyte technology. All molecules including the CEACAM5-TCB(P1AD5299) were used at 10 nM. FIG. 9A shows representative results fromone donor with technical triplicates. FIG. 9B shows the statisticalanalysis of technical triplicates expressed as area under the curve(AUC) at t=90 h of 1 donor in 1 experiment. Boxes display 25th-75thpercentiles, whiskers display min to max. Statistical analysis wasperformed by paired 1-way ANOVA. ***: p<0.001. It is shown thatCEA-targeted CD28 agonists with conventional CD28 agonistic binders donot behave superagonistically.

In FIGS. 10A, 10B and 10C it is shown that targeted CD28 agonists withmonovalent superagonistic binders are not functionally superagonistic.PBMC T cells were co-cultured for 5 days with 3T3-huFAP cells inpresence of increasing concentrations of FAP-CD28 with bivalent CD28binders (P1AD9011, closed circles) or FAP-CD28 with monovalency for CD28binding (P1AD4492, open circles). In FIG. 10A CFSE-dilution of CD8 Tcells is shown. Furthermore, activation of T cells was assessed bydetection of activation markers CD69 (FIG. 10B) and CD25 (FIG. 10C) byflow cytometry. Mean fluorescent intensity (MFI) of CD69 and CD25stainings are shown at 5 days post stimulation. Technical triplicatesfrom 1 donor are shown, error bars indicate SEM. It is shown thatTGN1412-like superagonism requires multivalent CD28 binding.

FIGS. 11A and 11B show that if combined with T cell bispecificantibodies (TCBs) TCB-mediated effector functions are supported bymonovalent and bivalent CD28 binding of FAP-targeted agonistic CD28antigen binding molecules with comparable potency, but CD28 bindermonovalency is required to maintain tumor target dependence of CD28agonists in the presence of TCBs. In FIG. 11A, for the presence of FAP,PBMC T cells were incubated with MCSP- and FAP-expressing MV3 melanomacells for 90 h in the presence of a combined limiting concentration ofMCSP-TCB (5 pM, P1AD2189) and increasing concentration (range 0-nM) ofFAP-CD28(SA) with bivalent or monovalent binding to CD28, respectively.Depicted is target cell killing at 90 h as assessed by live cell imagingusing the IncuCyte technology. In FIG. 11B, PBMC T cells wereco-cultured for 90 h with FAP-negative CEA-expressing MKN45 gastriccancer cells (FAP absent) in the presence of limiting concentrations ofCEACAM5-TCB (10 pM, P1AD5299) in combination and increasingconcentration (range 0-10 nM) of FAP-CD28 with bivalent or monovalentCD28 binding, respectively. Depicted is target cell killing at 90 h asassessed by IncuCyte. Data show killing of MKN45 target cells over time,from 1 donor in 1 experiment, technical triplicates, error bars indicateSEM.

In FIGS. 12A and 12B it is shown that FAP-CD28(SA) with bivalent bindingto CD28 loses FAP-dependence when combined with T cell bispecifics. InFIG. 12A no TCB is present. PBMC T cells were either co-cultured withCEA-expressing MKN45 and 3T3-huFAP (“FAP present” condition, closedcircles) or 3T3-WT (“FAP absent” condition, open circles), respectively,in the presence of increasing concentrations of FAP-CD28(SA) 2+1. Thecombination with TCB is shown in FIG. 12B. PBMC T cells were eitherco-cultured with CEA-expressing MKN45 and 3T3-huFAP (“FAP present”condition, closed circles) or 3T3-WT (“FAP absent” condition, opencircles), respectively, in the presence of limiting concentrations ofCEACAM5-TCB (10 pM, P1AD5299) and increasing concentrations of FAP-CD282+1 SA. Shown is CD8 T cell proliferation after 5 days of stimulation.Data are representative of 2 independent experiments with 2 donors.Results from one donor are shown, data points represent technicaltriplicates, error bars indicate SEM.

FIGS. 13A, 13B and 13C show the functionality of FAP-CD28(SA) antigenbinding molecules with monovalent binding to CD28 in different formats.Molecule C is a FAP-CD28(SA) classical 1+1 format (P1AD4492), Molecule His a FAP-CD28(SA) 1+1 “head-to-tail” (H2T) format (P1AE2021), Molecule Iis a FAP-CD28(SA) 1+1 format with C-terminal fusion of the FAP binder(P1AE2236) and Molecule G is a FAP-CD28(SA) 2+1 format (P1AD5231). Asreference the bivalent CD28 antigen binding molecule (P1AD9011) wasused. PBMC T cells were incubated with MCSP- and FAP-expressing MV3melanoma cells in the presence of limiting concentration of MCSP-TCB (5pM, P1AD2189) and increasing concentration (range 0-10 nM) of FAP-CD28in the given formats. Depicted is CF SE-dilution as measure for T cellproliferation of CD8 (FIG. 13A) and CD4 T cells (FIG. 13B) after 5 days,assessed by flow cytometry. FIG. 13C: Shown is killing of MV3 cells over84 hours in presence of 5 pM MCSP-TCB alone compared to combination of 5pM MCSP-TCB and increasing concentrations of FAP-CD28 in the variousformats. Killing was assessed by live cell imaging using the IncuCytesystem. All molecules were able to support TCB-mediated effectorfunctions. Graphs depict cumulative data from 3 independent experimentsand 4 donors 10 pM MCSP-TCB; E:T 20; Statistics: 2-way ANOVA. Starsindicate lowest concentration at which add-on is significant over TCBalone: *p<0.05, **p<0.01; ***p<0.001. Error bars indicate SEM.

Target cell killing of CEA-CD28 1+1 format in combination with TCB isshown in FIG. 14 . PBMC T cells were co-cultured for 90 h withCEA-expressing MKN45 gastric cancer cells in presence of limitingconcentrations of CEACAM5-TCB (10 pM, P1AD5299) in combination with 2 nMof CEA-CD28 (P1AE3127) or untargeted CD28 (P1AD8944). Data show killingof MKN45 target cells over time, from 1 donor in 1 experiment. Killingwas assessed by live cell imaging using the IncuCyte system. It is shownthat only the combination leads to target cell killing, at the givenconcentration the molecules alone do not induce killing. CEA-CD28synergizes with CEACAM5-TCB.

In FIG. 15 it is shown that CEA-CD28 enhances CEA-TCB and CEACAM5-TCBand lowers the threshold of CEA-expression for TCBs to induce T cellactivation. PBMC T cells were incubated with increasing concentrationsof either CEA-TCB (P1AD4646) or CEACAM5-TCB (P1AD5299) and fixedconcentrations of CEA-CD28 (P1AE3127) in presence of target cell lineswith different CEA expression levels: (i) MKN45 (high expression,approx. 400 000 CEA binding sites/cell), (ii) Lovo (medium expression,approx. 60 000 CEA binding sites/cell), (iii) HT-29 (low expression,approx. 6 000 CEA binding sites/cell). T cell proliferation was assessedas proxy of T cell activation by flow cytometry.

The binding of selected affinity reduced CD28 binder variants in abispecific CEA-targeted monovalent 1+1 format to CD28 on cells is shownin FIG. 16 . Median fluorescence intensities of binding of the CEA-CD28antibodies or anti-DP47 antibody (negative control) to CHO cellsexpressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61, modifiedto stably overexpress human CD28) was assessed by flow cytometry. Shownare technical triplicates with SEM.

FIGS. 17A, 17B and 17C show the functionality of selected affinityreduced CD28 binder variants in a bispecific CEA-targeted monovalent 1+1format. PBMC T cells were co-cultured with CEA-expressing MKN45 gastriccancer cells in presence of limiting concentrations of CEACAM5-TCB (10pM, P1AD5299) in combination with 2 nM of the CEA-CD28 1+1 moleculeswith the CD28 binder variants. CD8 T cell proliferation (FIG. 17A) andCD4 T cell proliferation (FIG. 17B) was assessed by CFSE-dilution byflow cytometry after 5 days of co-culture. Target cell killing wasassessed after 90 h of incubation (FIG. 17C). Data show killing of MKN45target cells over time, from 1 donor in 1 experiment. All molecules areable to support CEACAM5-TCB mediated effector functions.

FIG. 18 shows the binding of humanized CEA(A5B7) huIgG1 P329G LALAvariants to MKN-45 as compared to the binding of the parental murineA5B7 antibody. Antibodies were detected with a fluorescently labeledsecondary antibody and fluorescence was measured by flow cytometry.

FIGS. 19A to 19C are schematic illustrations of the recombinant proteinsdisplaying different domains of the CEACAM5 protein that were used asantigens in the phage display campaign. FIG. 19A shows constructNABA-avi-His consisting of the 4 Ig-like domains N, A1, B and A2. FIG.19B shows the construct N(A2B2)A-avi-His and FIG. 19C illustrates theconstruct NA(B2)A-avi-His.

FIGS. 20A and 20B show the VH and VL sequences, respectively, ofhumanized CEA antibody A5H1EL1D wherein the randomized positions aremarked with X.

Schematic drawings of the phage vectors of the affinity maturationlibraries are shown in FIG. 21A (CDRH1/H2 affinity maturation library),FIG. 21B (CDRL1/H2 affinity maturation library) and FIG. 21C(CDRH3/CDRL3 amplification library).

FIGS. 22A and 22B show an alignment of the VH amino acid sequences (FIG.22A) and VL amino acid sequences (FIG. 22B) of the affinity-matured,humanized CEA(A5H1EL1D) antibody variants.

In FIGS. 23A to 23D schematic illustrations of bispecific CEA/CD28antigen binding molecules as described in Example 11 are shown.

FIG. 23A shows a bispecific CEA-CD28 antigen binding molecule in 1+1format, wherein the CEA antigen binding domain is represented ascrossFab (VH/VL exchange) and in the Fab fragment bearing the CD28antigen binding domain there are charged modifications in order tosupport the correct pairing of the light chains. The Fc domain has knobinto hole modifications and the P329G LALA mutations to abrogate thebinding to Fey receptors. In FIG. 23B the CD28 antigen binding domain isrepresented as crossFab (VH/VL exchange) and the Fab fragment bearingthe CEA antigen binding domain comprises the charged modifications.

FIG. 23C illustrates a bispecific CEA-CD28 antigen binding molecule in2+1 format, wherein the CD28 antigen binding domain is represented ascrossFab and two Fab fragments with CEA antigen binding domains arefused to each other via the heavy chain (head-to tail).

FIG. 23D illustrates a bispecific CEA-CD28 antigen binding molecule in2+1 format, wherein the CD28 antigen binding domain is represented ascrossFab that is fused at its C-terminus to the N-terminus of one of theheavy chains of the “bivalent” CEA antibody (“classical” format).

In FIG. 24 it is shown that affinity-matured anti-CEA clone P002.139shows improved binding to CEACAM5 on CEA-expressing MV3 cells. Shown isbinding of CEA-CD28 bispecific antibodies carrying either theaffinity-matured anti-CEA clone P002.139 or the parental A5H1EL1D clone.Median fluorescence intensities of binding of the CEA-CD28 bispecificantibodies or anti-DP47 antibody (negative control) to MV3 cellsgenetically engineered to express human CEACAM5 was assessed by flowcytometry. Shown are technical duplicates with SEM. Graph isrepresentative of 3 independent experiments.

In FIGS. 25A and 25B it is shown that affinity-matured anti-CEA cloneP002.139 shows improved functionality in an IL-2 reporter assay. Shownis luminescence readout after 6 h of co-incubation of MKN45 cells, IL-2reporter cells with 5 nM CEA-TCB and CEA-CD28 carrying either theaffinity-matured clone P002.139 or the parental clone A5H1EL1D. FIG. 25Ashows dose response. Dotted line indicates luminescence achieved byCEA-TCB alone. In FIG. 25B the Area under the curve values calculatedfrom data depicted in FIG. 25A are shown. Provided are technicalduplicates with SEM. Graph is representative of 3 independentexperiments.

FIG. 26 shows the study design of an efficacy study with bispecificCEA-CD28 antibodies (comparison of different CEA clones) in combinationwith CEA TCB in MKN45 Xenograft in humanized mice. Shown is the designand the different treatment groups.

FIGS. 27A to 27E show results of the efficacy study with CEA-CD28 andCEA TCB combination in MKN45 Xenograft in humanized mice. Shown is theaverage tumor volume (FIG. 27A) or the growth of tumors in individualmice for the four treatment groups as plotted on the y-axis (FIGS. 27Bto 27E). FIG. 27B shows the tumor growth for each individual mouse inthe vehicle group, FIG. 27C of the mice treated with CEA TCB alone, FIG.27D of mice treated with CEA TCB and CEA(T84.66)-CD28 (SA_Variant 15)and FIG. 27E of mice treated with CEA TCB and CEA(A5H1EL1D)-CD28(SA_Variant 15). It can be seen that there is increased TCB-mediatedtumor regression in the presence of both bispecific CEA-CD28 antibodies.

FIG. 28 shows the study design of an efficacy study with bispecificCEA-CD28 antibodies (comparison of different CD28 clones) in combinationwith CEACAM5 TCB in BXPC3 Xenograft in humanized mice. Shown is thedesign and the different treatment groups.

FIG. 29 shows the tumor growth kinetics (Mean, +SEM) for all treatmentgroups, the corresponding TGI values of each treatment arm are shown inTable 33 (Example 13.2).

The ex vivo Immuno-PD data are shown in FIGS. 30A to 30D. FIG. 30A showsrepresentative dot plots (CD3 against CD45 and CD4 against CD8) of thestained tumor single cell suspensions of each treatment arm. The summaryof CD3, CD8 and CD4 T cell infiltration is depicted in FIG. 30B (CD3),FIG. 30C (CD8) and FIG. 30D (CD4), respectively.

FIG. 31 shows the study design of an efficacy study with bispecificCEA-CD28 antibody (CEA(A5H1EL1D)-CD28 (SA_Variant 8)) in combinationwith CEA TCB in MKN45 Xenograft in humanized mice. Shown is the designand the different treatment groups.

FIG. 32 shows the tumor growth kinetics (Mean, +SEM) for all treatmentgroups, the corresponding TGI values of each treatment arm are shown inTable 35 (Example 13.3).

The ex vivo Immuno-PD data are shown in FIGS. 33A and 33B. FIG. 33Ashows representative dot plots of the stained tumor single cellsuspensions of each treatment arm. The summary of CD3+ T cellinfiltration is depicted in FIG. 33B.

In FIGS. 34A to 34D schematic illustrations of bispecific CD28 antigenbinding molecules as described in Example 14 are shown.

FIG. 34A shows a bispecific EpCAM-CD28 antigen binding molecule in 1+1format, wherein the CD28 antigen binding domain is represented ascrossFab (VH/VL exchange) and in the Fab fragment bearing the EpCAMantigen binding domain there are charged modifications in order tosupport the correct pairing of the light chains. The Fc domain has knobinto hole modifications and the P329G LALA mutations to abrogate thebinding to Fcγ receptors.

In FIG. 34B the Fab bearing the CD28 antigen binding domain comprisescharged modifications and the Fab bearing the HER3 antigen bindingdomain is represented as crossFab (VH/VL exchange).

FIG. 34C illustrates a bispecific CD30-CD28 antigen binding molecule in1+1 format, wherein Fab molecule bearing the CD28 antigen binding domaincomprises charged modifications and the Fab bearing the CD30 antigenbinding domain is represented as crossFab (VH/VL exchange).

FIG. 34D illustrates a bispecific TPBG-CD28 antigen binding molecule in1+1 format, wherein Fab molecule bearing the CD28 antigen binding domaincomprises charged modifications and the Fab bearing the TPBG (5T4)antigen binding domain is represented as crossFab (VH/VL exchange).

FIGS. 35A to 35C relate to the functional characterization of EpCAM-CD28bispecific antigen binding molecules. In FIG. 35A it is shown thatEpCAM-CD28 (Molecule 14A) binds to human CD28 on CHO-k1 cells expressingCD28, assessed by flow cytometry. The binding to EpCAM on HT29 cells,assessed by flow cytometry, is shown in FIG. 35B. Anti-DP47 served asnegative control for unspecific binding of antibody compounds to cells.Dots represent means of technical duplicates. In FIG. 35C it is shownthat EpCAM-CD28 (P1AE9051) enhances T cell responses to anti-CD3stimulus in the IL-2 reporter assay. Shown is IL-2 reporter cellactivation measured by luminescence readout after 6 hours ofco-incubation with HT-29 in presence of suboptimal concentrations ofanti-CD3 IgG (10 nM) and increasing concentrations of EpCAM-CD28. Dotsrepresent means of technical duplicates.

FIGS. 36A to 36C relate to the functional characterization of HER3-CD28bispecific antigen binding molecules. In FIG. 36A it is shown thatHER3-CD28 (P1AF0151) binds to human CD28 on CHO-k1 cells expressingCD28, assessed by flow cytometry. The binding to of HER3-CD28 to HER3 onT-47D cells, assessed by flow cytometry, is shown in FIG. 36B. Anti-DP47served as negative control for unspecific binding of antibody compoundsto cells. Dots are means of technical duplicates. In FIG. 36C it isshown that HER3-CD28 (P1AF0151) enhances T cell responses to anti-CD3stimulus in the IL-2 reporter assay. Shown is IL-2 reporter cellactivation measured by luminescence readout after 6 hours ofco-incubation with T-47D cells in presence of suboptimal concentrationsof anti-CD3 IgG clone OKT3 (10 nM) and increasing concentrations ofHER3-CD28. Dots represent means of technical duplicates.

In FIGS. 37A to 37C schematic illustrations of bispecific CD28 antigenbinding molecules targeting a Multiple Myeloma (MM) cell surface antigenas described in Example 16 are shown.

FIG. 37A shows a bispecific GPRC5D-CD28 antigen binding molecule in 1+1format, wherein the CD28 antigen binding domain is represented ascrossFab (VH/VL exchange) and in the Fab fragment bearing the CPRC5Dantigen binding domain there are charged modifications in order tosupport the correct pairing of the light chains. The Fc domain has knobinto hole modifications and the P329G LALA mutations to abrogate thebinding to Fcγ receptors. In FIG. 37B the Fab bearing the CD28 antigenbinding domain comprises charged modifications and the Fab bearing theCD38 antigen binding domain is represented as crossFab (VH/VL exchange).

FIG. 37C illustrates a bispecific BCMA-CD28 antigen binding molecule in1+1 format, wherein Fab molecule bearing the CD28 antigen binding domainis represented as crossFab (VH/VL exchange) and the Fab bearing the BCMAantigen binding domain comprises charged modifications.

FIG. 37D illustrates the anti-GPRC5D/anti-CD3 bispecific antibody(GPRC5D TCB) in 2+1 format, wherein the Fab molecules bearing the GPCR5Dantigen binding domain comprise charged modifications and the Fabbearing the CD3 antigen binding domain is represented as crossFab (VH/VLexchange).

FIGS. 38A to 38F relate to the binding of bispecific antigen bindingmolecules targeting CD28 and a Multiple Myeloma (MM) cell surfaceantigen to cells (Example 17.1). Shown is the binding of bispecificantigen binding molecules to either human CD28 on CHO huCD28 c145 cells(FIGS. 38A and 38E), to human CD38 on OCI-Ly18 cells (FIG. 38B), humanBCMA (B-cell maturation antigen, FIG. 38C) on IM-9 cells and to humanGPRC5D on CHO huGPRC5D L2 cells (FIGS. 38D and 38F) expressed on theindicated cell lines. Depicted are relative median fluorescence valus(MFI) from duplicates with SD. EC₅₀ values of binding were calculated byGraphPadPrism and are included in Table 38.

The T-cell activation of bispecific antigen binding molecules targetingCD28 and a Multiple Myeloma (MM) cell surface antigen as assessed in theIL-2 reporter assay is shown in FIGS. 39A to 39F. Shown is theIL2-reporter cell assay after 5 and 22 hours of incubation, asdetermined by luminescence. IL2-reporter effector and GPRC5D-expressingtarget cells were incubated at a effector to target ratio (E:T) of 5:1.GPRC5D-TCB was added at a fixed final assay concentration of 1 nM, theindicated MM-targeted CD28 bispecific antigen binding molecules weretitrated as indicated. Representative dose-response curves are depictedfor CD38-CD28 in FIG. 39A (after 5 hours of incubation) and FIG. 39B(after 22 hours), for BCMA-CD28 in FIG. 39C (after 5 hours) and FIG. 39D(after 22 hours) and for GPRC5D-CD28 in FIG. 39E (after 5 hours) andFIG. 39F (after 22 hours of incubation).

FIGS. 40A to 40C show the boosting of T-cell mediated lysis of theGPRC5D-expressing MM cell line NCI-H929 in presence of 0.2 nM of theindicated CD28 bispecific molecules CD38-CD28 (FIG. 40A), BCMA-CD28(FIG. 40B) and GPRC5D-CD28 (FIG. 40C). Lysis was determined afterco-incubation of human pan T-cells and MM tumor target cells at a finalE:T ratio of 1:1 for 22 hours. Depicted are technical duplicates withSD. EC₅₀ values and area under the curve values of tumor cell lysis werecalculated by GraphPadPrism and are depicted in Table 39.

FIG. 41A shows a schematic illustration of a bispecific CD19-CD28antigen binding molecule in 1+1 format as described in Example 18,wherein in the Fab comprising the CD19 antigen binding domain the VH andVL domains are exchanged with each other (VH/VL crossfab) and wherein inthe Fab comprising the CD28 antigen binding domain certain amino acidsin the CH1 and CL domain are exchanged (charge variants) to allow betterpairing with the light chain. FIG. 41B shows a corresponding moleculewherein the CD19 antigen binding domain has been replaced by a CD79bantigen binding domain (anti-CD79b crossfab).

FIG. 42 relates to the determination of kinetic and thermodynamicparameters of CD79b (polatuzumab) in the construct CD79b(huMA79b.v28)-CD28 (v15) 1+1. Soluble recombinant CD79b-His was capturedon CM5 chip via an anti-penta-His antibody and bi-specific CD79b(huMA79b.v28)-CD28 (v15) 1+1 was used as an analyte. Smooth linesrepresent a global fit of the data to a 1:1 interaction mode.

In FIG. 43A the median fluorescence intensities (MFI) of binding ofCD19-CD28 variant 15 (P1AE9040) to four different B cell linesexpressing different levels of CD19 are shown. Binding was assessed byflow cytometry. Shown are technical duplicates with SEM. In FIG. 43BFACS staining for CD19 of the four different B cell lines (MFI) isdepicted.

Binding of CD19-CD28 with varying CD28 affinities to human CD19 and CD28on cells is shown in FIGS. 44A and 44B. Median fluorescence intensities(MFI) of binding to CHOk1-CD28 cells (FIG. 44A) and of binding to CD19on Nalm6 B cells (FIG. 44B) are shown. Dots represent technicalduplicates with SEM. Corresponding EC₅₀ values are shown in Table 42(CHOk1-CD28) and Table 43 (Nalm6) of Example 20. Binding was assessed byflow cytometry.

In FIGS. 45A to 45D it is shown that CD19-CD28v15 enhances CD20-TCB inIL-2 reporter assay in presence of different B cell lines. Shown is IL-2reporter cell activation measured by luminescence readout (LUM) after 6hours of co-incubation with different B cell lines in presence ofsuboptimal concentrations of CD20-TCB and increasing concentrations ofCD19-CD28v15. Dots represent technical duplicates with SEM. SuboptimalCD20-TCB concentration differs with target cell lines: 10 nM for Nalm6,0.05 nM for RCK8, WSU DLCL2 and Z138.

FIG. 46 illustrates that CD19-CD28 with various CD28 affinities enhancesCD20-TCB mediated T cell activation. Shown is IL-2 reporter cellactivation measured by luminescence readout (LUM) after 6 hours ofco-incubation with Nalm6 B cells in presence of suboptimalconcentrations of CD20-TCB (10 nM) and increasing concentrations ofCD19-CD28v15. Dots represent technical duplicates with SEM.

The activation status of PBMC-derived T cells after co-culture withCD20-expressing target cells (Nalm6) (E:T ratio 5:1) and CD19-CD28 inabsence or presence of CD20-TCB was assessed. The activity of CD19-CD28in absence or presence of TCR signals is shown in FIG. 47 . Shown isCD69 expression of PBMC-derived CD4 T cells after 48 h of co-incubationwith Nalm6 cells, increasing concentrations of CD19-CD28v15 in presenceor absence of 10 nM CD20-TCB. Dots represent technical triplicates withSEM.

In FIGS. 48A to 48D it is illustrated that CD19-CD28 alone does notinduce cytokine secretion in PBMCs. Shown is cytokine release in wholePBMCs after 48 hours of co-culture with CD19-CD28 molecules in presenceor absence of CD20-TCB. Bars represent mean+SEM of technicaltriplicates. Data are representative of 2 donors. Cytokine secretion wasassessed by Bio-Plex Pro Human Cytokine 17-plex Assay. Shown are IFNγ(FIG. 48A), IL-2 (FIG. 48B), IL-(FIG. 48C), and TNF (FIG. 48D).

In FIGS. 49A and 49B, functional data relating to CD79b-CD28 are shownenhances CD20-TCB in IL-2 reporter assay in presence of Z138 B cells. InFIG. 49A, median fluorescence intensities (MFI) of binding to CD79b onZ138 B cells is shown. In FIG. 49B it is shown that CD79b-CD28 enhancesCD20-TCB in IL-2 reporter assay in presence of Z138 B cells. Shown isIL-2 reporter cell activation measured by luminescence readout (LUM)after 6 hours of co-incubation with different B cell lines in presenceof suboptimal concentrations of CD20-TCB and increasing concentrationsof CD79b-CD28. Dots represent technical duplicates with SEM.

FIG. 50 shows the study design of an efficacy study with bispecificCD19-CD28 antibodies (comparison of two different CD28 clones) in NALM6Xenograft in humanized mice. Shown is the design and the differenttreatment groups.

FIGS. 51A to 51D show results of the efficacy study with CD19-CD28 inNALM6 Xenograft in humanized mice. Shown is the average tumor volume(FIG. 51A) or the growth of tumors in individual mice for the threetreatment groups as plotted on the y-axis (FIG. 51B to 51D). FIG. 51Bshows the tumor growth for each individual mouse in the vehicle group,FIG. 51C of the mice treated with CD19-CD28 (variant 15) and FIG. 51D ofmice treated with CD19-CD28 (variant 8). It can be seen that CD19-CD28(variant 8) as a single agent induced stronger tumor growth inhibitionas compared to CD19-CD28 (variant 15).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as generally used in the art to which thisinvention belongs. For purposes of interpreting this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are antibodies,multispecific antibodies (e.g., bispecific antibodies), antibodyfragments and scaffold antigen binding proteins.

As used herein, the term “antigen binding domain that binds to atumor-associated antigen” or “moiety capable of specific binding to atumor-associated antigen” refers to a polypeptide molecule thatspecifically binds to an antigenic determinant. In one aspect, theantigen binding domain is able to activate signaling through its targetcell antigen. In a particular aspect, the antigen binding domain is ableto direct the entity to which it is attached (e.g. the CD28 antibody) toa target site, for example to a specific type of tumor cell or tumorstroma bearing the antigenic determinant. Antigen binding domainscapable of specific binding to a target cell antigen include antibodiesand fragments thereof as further defined herein. In addition, antigenbinding domains capable of specific binding to a target cell antigeninclude scaffold antigen binding proteins as further defined herein,e.g. binding domains which are based on designed repeat proteins ordesigned repeat domains (see e.g. WO 2002/020565).

In relation to an antigen binding molecule, i.e. an antibody or fragmentthereof, the term “antigen binding domain that binds to a target cellantigen” refers to the part of the molecule that comprises the areawhich specifically binds to and is complementary to part or all of anantigen. An antigen binding domain capable of specific antigen bindingmay be provided, for example, by one or more antibody variable domains(also called antibody variable regions). Particularly, an antigenbinding domain capable of specific antigen binding comprises an antibodylight chain variable region (VL) and an antibody heavy chain variableregion (VH). In another aspect, the “antigen binding domain capable ofspecific binding to a tumor-associated antigen” can also be a Fabfragment or a crossFab fragment. In another aspect, the “antigen bindingdomain capable of specific binding to a tumor-associated antigen” canalso be a Fab fragment or a crossFab fragment. As used herein, the terms“first”, “second” or “third” with respect to antigen binding domainsetc., are used for convenience of distinguishing when there is more thanone of each type of moiety. Use of these terms is not intended to confera specific order or orientation of the moiety unless explicitly sostated.

As used herein, the term “antigen binding domain that binds to a B cellsurface antigen” or “moiety capable of specific binding to a B cellsurface antigen” refers to a polypeptide molecule that specificallybinds to an antigenic determinant on the B cell surface. In one aspect,the antigen binding domain is able to activate signaling through itstarget cell antigen. In a particular aspect, the antigen binding domainis able to direct the entity to which it is attached (e.g. the CD28agonist) to a target site, e.g. on the B cell. Antigen binding domainscapable of specific binding to a B cell surface antigen includeantibodies and fragments thereof as further defined herein. In addition,antigen binding domains capable of specific binding to a B cell surfaceantigen include scaffold antigen binding proteins as further definedherein, e.g. binding domains which are based on designed repeat proteinsor designed repeat domains (see e.g. WO 2002/020565).

The term “antigen binding domain that binds to a Multiple Myeloma (MM)cell surface antigen” or “moiety capable of specific binding to aMultiple Myeloma (MM) cell surface antigen” refers to a polypeptidemolecule that specifically binds to an antigenic determinant on MultipleMyeloma (MM) cell. In one aspect, the antigen binding domain is able toactivate signaling through its target cell antigen. In a particularaspect, the antigen binding domain is able to direct the entity to whichit is attached (e.g. the CD28 agonist) to a target site, e.g. on the MMcell. Antigen binding domains capable of specific binding to a MultipleMyeloma (MM) cell surface antigen include antibodies and fragmentsthereof as further defined herein. In addition, antigen binding domainscapable of specific binding to a B cell surface antigen include scaffoldantigen binding proteins as further defined herein, e.g. binding domainswhich are based on designed repeat proteins or designed repeat domains(see e.g. WO 2002/020565).

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, monospecific and multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen. The term “bispecific” means that the antigen bindingmolecule is able to specifically bind to at least two distinct antigenicdeterminants. Typically, a bispecific antigen binding molecule comprisestwo antigen binding sites, each of which is specific for a differentantigenic determinant. However, a bispecific antigen binding moleculemay also comprise additional antigen binding sites which bind to furtherantigenic determinants. In certain aspects, the bispecific antigenbinding molecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells or on the same cell. The term “bispecific” in accordancewith the present invention thus may also include a trispecific molecule,e.g. a bispecific molecule comprising a CD28 antibody and two antigenbinding domains directed to two different target cell antigens.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites specific for onedistinct antigenic determinant in an antigen binding molecule that arespecific for one distinct antigenic determinant. As such, the terms“bivalent”, “tetravalent”, and “hexavalent” denote the presence of twobinding sites, four binding sites, and six binding sites specific for acertain antigenic determinant, respectively, in an antigen bindingmolecule. In particular aspects of the invention, the bispecific antigenbinding molecules according to the invention can be monovalent for acertain antigenic determinant, meaning that they have only one bindingsite for said antigenic determinant or they can be bivalent ortetravalent for a certain antigenic determinant, meaning that they havetwo binding sites or four binding sites, respectively, for saidantigenic determinant.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called a (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2),γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, triabodies, tetrabodies, crossFab fragments; linearantibodies; single-chain antibody molecules (e.g. scFv); and singledomain antibodies. For a review of certain antibody fragments, seeHudson et al., Nat Med 9, 129-134 (2003). For a review of scFvfragments, see e.g. Plückthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90, 6444-6448 (1993). Triabodies and tetrabodies are also describedin Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodiesare antibody fragments comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S.Pat. No. 6,248,516 B1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion of anintact antibody as well as production by recombinant host cells (e.g. E.coli or phage), as described herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains and also the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.As used herein, Thus, the term “Fab fragment” refers to an antibodyfragment comprising a light chain fragment comprising a variable lightchain (VL) domain and a constant domain of a light chain (CL), and avariable heavy chain (VH) domain and a first constant domain (CH1) of aheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxy terminus of the heavy chain CH1 domainincluding one or more cysteins from the antibody hinge region. Fab′-SHare Fab′ fragments in which the cysteine residue(s) of the constantdomains bear a free thiol group. Pepsin treatment yields an F(ab′) 2fragment that has two antigen-combining sites (two Fab fragments) and apart of the Fc region.

The term “crossFab fragment” or “xFab fragment” or “crossover Fabfragment” refers to a Fab fragment, wherein either the variable regionsor the constant regions of the heavy and light chain are exchanged. Twodifferent chain compositions of a crossover Fab molecule are possibleand comprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable (VL) domain and the heavy chainconstant domain (CH1), and a peptide chain composed of the heavy chainvariable domain (VH) and the light chain constant domain (CL). Thiscrossover Fab molecule is also referred to as CrossFab_((VLVH)). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable domain (VH) and the light chainconstant domain (CL), and a peptide chain composed of the light chainvariable domain (VL) and the heavy chain constant domain (CH1). Thiscrossover Fab molecule is also referred to as CrossFab_((CLCH1)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabfragments are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g. position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is apolypeptide consisting of an antibody heavy chain variable domain (VH),an antibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 andb) VL-CH1-linker-VH-CL; wherein VH and VL form together anantigen-binding site which binds specifically to an antigen and whereinsaid linker is a polypeptide of at least 30 amino acids. In addition,these x-scFab molecules might be further stabilized by generation ofinterchain disulfide bonds via insertion of cysteine residues (e.g.position 44 in the variable heavy chain and position 100 in the variablelight chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of thevariable regions of the heavy (V_(H)) and light chains (V_(L)) of anantibody, connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the VH with the C-terminus of the VL, or vice versa. Thisprotein retains the specificity of the original antibody, despiteremoval of the constant regions and the introduction of the linker. scFvantibodies are, e.g. described in Houston, J. S., Methods in Enzymol.203 (1991) 46-96). In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property of fulllength antibodies.

“Scaffold antigen binding proteins” are known in the art, for example,fibronectin and designed ankyrin repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Engineered protein scaffolds as next-generationantibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumppet al., Darpins: A new generation of protein therapeutics. DrugDiscovery Today 13: 695-701 (2008). In one aspect of the invention, ascaffold antigen binding protein is selected from the group consistingof CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derivedmolecule such as Z-domain of Protein A (Affibody), an A-domain(Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrinrepeat protein (DARPin), a variable domain of antibody light chain orheavy chain (single-domain antibody, sdAb), a variable domain ofantibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domain of anew antigen receptor beta-lactamase (V_(NAR) fragments), a humangamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domainof human protease inhibitors, microbodies such as the proteins from theknottin family, peptide aptamers and fibronectin (adnectin). CTLA-4(Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptorexpressed on mainly CD4⁺ T-cells. Its extracellular domain has avariable domain-like Ig fold. Loops corresponding to CDRs of antibodiescan be substituted with heterologous sequence to confer differentbinding properties. CTLA-4 molecules engineered to have differentbinding specificities are also known as Evibodies (e.g. U.S. Pat. No.7,166,697B1). Evibodies are around the same size as the isolatedvariable region of an antibody (e.g. a domain antibody). For furtherdetails, see Journal of Immunological Methods 248 (1-2), 31-45 (2001).Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid beta-sheet secondary structure with a number of loopsat the open end of the conical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details, seeBiochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 andUS20070224633. An affibody is a scaffold derived from Protein A ofStaphylococcus aureus which can be engineered to bind to antigen. Thedomain consists of a three-helical bundle of approximately 58 aminoacids. Libraries have been generated by randomization of surfaceresidues. For further details, see Protein Eng. Des. Sel. 2004, 17,455-462 and EP 1641818A1. Avimers are multidomain proteins derived fromthe A-domain scaffold family. The native domains of approximately 35amino acids adopt a defined disulfide bonded structure. Diversity isgenerated by shuffling of the natural variation exhibited by the familyof A-domains. For further details, see Nature Biotechnology 23(12),1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),909-917 (June 2007). A transferrin is a monomeric serum transportglycoprotein. Transferrins can be engineered to bind different targetantigens by insertion of peptide sequences in a permissive surface loop.Examples of engineered transferrin scaffolds include the Trans-body. Forfurther details, see J. Biol. Chem 274, 24066-24073 (1999). DesignedAnkyrin Repeat Proteins (DARPins) are derived from Ankyrin which is afamily of proteins that mediate attachment of integral membrane proteinsto the cytoskeleton. A single ankyrin repeat is a 33 residue motifconsisting of two alpha-helices and a beta-turn. They can be engineeredto bind different target antigens by randomizing residues in the firstalpha-helix and a beta-turn of each repeat. Their binding interface canbe increased by increasing the number of modules (a method of affinitymaturation). For further details, see J. Mol. Biol. 332, 489-503 (2003),PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007)and US20040132028A1. A single-domain antibody is an antibody fragmentconsisting of a single monomeric variable antibody domain. The firstsingle domains were derived from the variable domain of the antibodyheavy chain from camelids (nanobodies or V_(H)H fragments). Furthermore,the term single-domain antibody includes an autonomous human heavy chainvariable domain (aVH) or V_(NAR) fragments derived from sharks.Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins consists of a backbone of the natural amino acid sequence ofthe 10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the beta-sandwich can be engineered toenable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details, see Protein Eng. Des. Sel. 18, 435-444(2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.Peptide aptamers are combinatorial recognition molecules that consist ofa constant scaffold protein, typically thioredoxin (TrxA) which containsa constrained variable peptide loop inserted at the active site. Forfurther details, see Expert Opin. Biol. Ther. 5, 783-797 (2005).Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can be engineered to include up to 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as areference molecule refers to an antigen binding molecule that blocksbinding of the reference molecule to its antigen in a competition assayby 50% or more, and conversely, the reference molecule blocks binding ofthe antigen binding molecule to its antigen in a competition assay by50% or more.

The term “antigen binding domain” refers to the part of an antigenbinding molecule that comprises the area which specifically binds to andis complementary to part or all of an antigen. Where an antigen islarge, an antigen binding molecule may only bind to a particular part ofthe antigen, which part is termed an epitope. An antigen binding domainmay be provided by, for example, one or more variable domains (alsocalled variable regions). Preferably, an antigen binding domaincomprises an antibody light chain variable domain (VL) and an antibodyheavy chain variable domain (VH).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins useful as antigens herein canbe any native form the proteins from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g. mice and rats),unless otherwise indicated. In a particular embodiment the antigen is ahuman protein. Where reference is made to a specific protein herein, theterm encompasses the “full-length”, unprocessed protein as well as anyform of the protein that results from processing in the cell. The termalso encompasses naturally occurring variants of the protein, e.g.splice variants or allelic variants.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding molecule to bind to aspecific antigen can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed ona BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)),and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).In one embodiment, the extent of binding of an antigen binding moleculeto an unrelated protein is less than about 10% of the binding of theantigen binding molecule to the antigen as measured, e.g. by SPR. Incertain embodiments, an molecule that binds to the antigen has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³M, e.g. from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (Kd), which is the ratio of dissociation andassociation rate constants (koff and kon, respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

A “tumor-associated antigen” or TAA as used herein refers to anantigenic determinant presented on the surface of a target cell, forexample a cell in a tumor such as a cancer cell, a cell of the tumorstroma, a malignant B lymphocyte or a melanoma cell. In certain aspects,the target cell antigen is an antigen on the surface of a tumor cell. Inone aspect, TAA is selected from the group consisting of FibroblastActivation Protein (FAP), Carcinoembryonic Antigen (CEA), Folatereceptor alpha (FolR1), Melanoma-associated Chondroitin SulfateProteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), humanepidermal growth factor receptor 2 (HER2), p95HER2, EpCAM, HER3, CD30 orTPBG (5T4), CD19, CD79b, CD20, CD22, CD37, CD38, BCMA and GPRC5D. In oneparticular aspect, TAA is selected from the group consisting ofFibroblast Activation Protein (FAP), Carcinoembryonic Antigen (CEA),Folate receptor alpha (FolR1), Melanoma-associated Chondroitin SulfateProteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), humanepidermal growth factor receptor 2 (HER2) and p95HER2. In anotherparticular aspect, TAA is selected from the group consisting ofFibroblast Activation Protein (FAP), Carcinoembryonic Antigen (CEA),EpCAM, HER3, CD30 or TPBG (5T4). In one particular aspect, thetumor-associated antigen is Fibroblast Activation Protein (FAP) orCarcinoembryonic Antigen (CEA). In one aspect, TAA is a B cell surfaceantigen selected from the group consisting of CD19, CD79b, CD20, CD22and CD37, in particular CD19 and CD79b. In one aspect, TAA is a MultipleMyeloma (MM) cell surface antigen selected from the group consisting ofCD38, BCMA and GPRC5D.

The term “Fibroblast activation protein (FAP)”, also known as Prolylendopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP fromany vertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed FAP as well as any form of FAP that results from processingin the cell. The term also encompasses naturally occurring variants ofFAP, e.g., splice variants or allelic variants. In one embodiment, theantigen binding molecule of the invention is capable of specific bindingto human, mouse and/or cynomolgus FAP. The amino acid sequence of humanFAP is shown in UniProt (world wide web.uniprot.org) accession no.Q12884 (version 149, SEQ ID NO:2), or NCBI (world wideweb.ncbi.nlm.nih.gov/) RefSeq NP 004451.2. The extracellular domain(ECD) of human FAP extends from amino acid position 26 to 760. The aminoacid sequence of a His-tagged human FAP ECD is shown in SEQ ID NO:135.The amino acid sequence of mouse FAP is shown in UniProt accession no.P97321 (version 126, SEQ ID NO:136), or NCBI RefSeq NP 032012.1. Theextracellular domain (ECD) of mouse FAP extends from amino acid position26 to 761. SEQ ID NO:137 shows the amino acid sequence of a His-taggedmouse FAP ECD. SEQ ID NO 138 shows the amino acid sequence, of aHis-tagged cynomolgus FAP ECD. Preferably, an anti-FAP binding moleculeof the invention binds to the extracellular domain of FAP.

The term “Carcinoembroynic antigen (CEA)”, also known asCarcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5),refers to any native CEA from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), unless otherwise indicated.The amino acid sequence of human CEA is shown in UniProt accession no.P06731 (version 151, SEQ ID NO:3). CEA has long been identified as atumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462,1965; Berinstein N. L., J Clin Oncol., 20:2197-2207, 2002). Originallyclassified as a protein expressed only in fetal tissue, CEA has now beenidentified in several normal adult tissues. These tissues are primarilyepithelial in origin, including cells of the gastrointestinal,respiratory, and urogential tracts, and cells of colon, cervix, sweatglands, and prostate (Nap et al., Tumour Biol., 9(2-3):145-53, 1988; Napet al., Cancer Res., 52(8):2329-23339, 1992). Tumors of epithelialorigin, as well as their metastases, contain CEA as a tumor associatedantigen. While the presence of CEA itself does not indicatetransformation to a cancerous cell, the distribution of CEA isindicative. In normal tissue, CEA is generally expressed on the apicalsurface of the cell (Hammarström S., Semin Cancer Biol. 9(2):67-81(1999)), making it inaccessible to antibody in the blood stream. Incontrast to normal tissue, CEA tends to be expressed over the entiresurface of cancerous cells (Hammarström S., Semin Cancer Biol.9(2):67-81 (1999)). This change of expression pattern makes CEAaccessible to antibody binding in cancerous cells. In addition, CEAexpression increases in cancerous cells. Furthermore, increased CEAexpression promotes increased intercellular adhesions, which may lead tometastasis (Marshall J., Semin Oncol., 30 (a Suppl. 8):30-6, 2003). Theprevalence of CEA expression in various tumor entities is generally veryhigh. In concordance with published data, own analyses performed intissue samples confirmed its high prevalence, with approximately 95% incolorectal carcinoma (CRC), 90% in pancreatic cancer, 80% in gastriccancer, 60% in non-small cell lung cancer (NSCLC, where it isco-expressed with HER3), and 40% in breast cancer; low expression wasfound in small cell lung cancer and glioblastoma.

CEA is readily cleaved from the cell surface and shed into the bloodstream from tumors, either directly or via the lymphatics. Because ofthis property, the level of serum CEA has been used as a clinical markerfor diagnosis of cancers and screening for recurrence of cancers,particularly colorectal cancer (Goldenberg D M., The InternationalJournal of Biological Markers, 7:183-188, 1992; Chau I., et al., J ClinOncol., 22:1420-1429, 2004; Flamini et al., Clin Cancer Res;12(23):6985-6988, 2006).

The term “epithelial cell adhesion molecule (EpCAM)” refers to anynative EpCAM from any vertebrate source, including mammals such asprimates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed EpCAM as well as any form ofEpCAM that results from processing in the cell. The term alsoencompasses naturally occurring variants of EpCAM, e.g., splice variantsor allelic variants. In one embodiment, the antigen binding molecule ofthe invention is capable of specific binding to human, mouse and/orcynomolgus EpCAM. The amino acid sequence of human EpCAM is shown inUniProt (world wide web.uniprot.org) accession no. P16422 (version 167,SEQ ID NO:68), or NCBI (world wide web.ncbi.nlm.nih.gov/) RefSeqNP_002345.2. The amino acid sequence of mouse EpCAM is shown in UniProt(world wide web.uniprot.org) accession no. Q99JW5 (version 111, SEQ IDNO:75), or NCBI (world wide web.ncbi.nlm.nih.gov/) RefSeq NP_032558.2.Epithelial cell adhesion molecule (EpCAM)—also known as tumor-associatedcalcium signal transducer 1 (TACSTD1), 17-1A and CD326—is a type I˜40kDa transmembrane glycoprotein that is frequently overexpressed incancers of epithelial origin and by cancer stem cells, and is thereforea molecule of significant interest for therapy and diagnosis. Theextracellular domain EpCAM can be cleaved to yield the solubleextracellular domain molecule EpEX, and the intracellular moleculeEpICD. EpICD has been shown to associate with other proteins to form anuclear complex which upregulates the expression of genes promoting cellproliferation. EpCAM may also be involved in the epithelial tomesenchymal cell transition (EMT), and may contribute to the formationof large metastases.

“CD30” or “TNFRSF8” is a member of the tumor necrosis factor receptorsuperfamily. It is characteristically expressed in certain hematopoieticmalignancies, including anaplastic large cell lymphoma and Hodgkinlymphoma, among others. The variable expression of CD30 on both normaland malignant lymphoid cells has focused research efforts onunderstanding the pathogenesis of CD30 upregulation, its contribution tolymphomagenesis through anti-apoptotic mechanisms, and its effect oncell survival. Given the restriction of CD30 to certain tumor types, thelogical extension of this has been to attempt to exploit it as atherapeutic target. The CD30 is a 120 kD transmembrane glycoproteinreceptor belonging to the tumor necrosis factor receptor (TNFR)superfamily, with intracellular, trans-membrane and extracellulardomains and the amino acid sequence of human CD30 is shown in UniProtaccession no. P28908 (SEQ ID NO:472).

The term “TPBG” refers to Trophoblast glycoprotein, also referred to as“5T4”. TBPG is a leucine-rich transmembrane glycoprotein involved incell adhesion. In adults this protein is highly expressed in many tumorcells and is associated with poor clinical outcome in numerous cancers.It refers to any native TPBG from any vertebrate source, includingmammals such as primates (e.g. humans) non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The amino acid sequence of human TPBG is shown in UniProtaccession no. Q13641 (SEQ ID NO: 473).

The term “FolR1” refers to Folate receptor alpha and has been identifiedas a potential prognostic and therapeutic target in a number of cancers.It refers to any native FolR1 from any vertebrate source, includingmammals such as primates (e.g. humans) non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The amino acid sequence of human FolR1 is shown in UniProtaccession no. P15328 (SEQ ID NO: 139), murine FolR1 has the amino acidsequence of UniProt accession no. P35846 (SEQ ID NO:140) and cynomolgusFolR1 has the amino acid sequence as shown in UniProt accession no.G7PR14 (SEQ ID NO:141). FolR1 is an N-glycosylated protein expressed onplasma membrane of cells. Foal has a high affinity for folic acid andfor several reduced folic acid derivatives and mediates delivery of thephysiological folate, 5-methyltetrahydrofolate, to the interior ofcells. FOLR1 is a desirable target for FOLR1-directed cancer therapy asit is overexpressed in vast majority of ovarian cancers, as well as inmany uterine, endometrial, pancreatic, renal, lung, and breast cancers,while the expression of FOLR1 on normal tissues is restricted to theapical membrane of epithelial cells in the kidney proximal tubules,alveolar pneumocytes of the lung, bladder, testes, choroid plexus, andthyroid, Recent studies have identified that FolR1 expression isparticularly high in triple negative breast cancers (Necela et al. PloSOne 2015, 10(3), e0127133).

The term “Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)”,also known as Chondroitin Sulfate Proteoglycan 4 (CSPG4) refers to anynative MCSP from any vertebrate source, including mammals such asprimates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The amino acidsequence of human MCSP is shown in UniProt accession no. Q6UVK1 (version103, SEQ ID NO:142). MCSP is a highly glycosylated integral membranechondroitin sulfate proteoglycan consisting of an N-linked 280 kDa.glycoprotein component and a 450-kDa. chondroitin sulfate proteoglycancomponent expressed on the cell membrane (Ross et al., Arch. Biochem,Biophys. 1983, 225:370-38). MCSP is more broadly distributed in a numberof normal and transformed cells. In particular, MCSP is found in almostall basal cells of the epidermis. MCSP is differentially expressed inmelanoma cells, and was found to be expressed in more than 90% of benignnevi and melanoma lesions analyzed. MCSP has also been found to beexpressed in tumors of nonmelanocytic origin, including basal cellcarcinoma, various tumors of neural crest origin, and in breastcarcinomas.

The term “Epidermal Growth Factor Receptor (EGFR)”, also namedProto-oncogene c-ErbB-1 or Receptor tyrosine-protein kinase erbB-1,refers to any native EGFR from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), unless otherwise indicated.The amino acid sequence of human EGFR is shown in UniProt accession no.P00533 (version 211, SEQ ID NO:143). The proto-oncogene “HER2”, (humanepidermal growth factor receptor 2) encodes a protein tyrosine kinase(p185HER2) that is related to and somewhat homologous to the humanepidermal growth factor receptor. HER2 is also known in the field asc-erbB-2, and sometimes by the name of the rat homolog, net.Amplification and/or overexpression of HER2 is associated with multiplehuman malignancies and appears to be integrally involved in progressionof 25-30% of human breast and ovarian cancers. Furthermore, the extentof amplification is inversely correlated with the observed medianpatient survival time (Slarnon, D. J. et al., Science 244:707-712(1989)). The amino acid sequence of human HER2 is shown in UniProtaccession no. P04626 (version 230, SEQ ID NO:144). The term “p95HER2” asused herein refers to a carboxy terminal fragment (CTF) of the HER2receptor protein, which is also known as “611-CTF” or “100-115 kDap95HER2”. The p95HER2 fragment is generated in the cell throughinitiation of translation of the HER2 mRNA at codon position 611 of thefull-length HER2 molecule (Anido et al, EMBO J 25; 3234-44 (2006)). Ithas a molecular weight of 100 to 115 kDa and is expressed at the cellmembrane, where it can form homodimers maintained by intermoleculardisulfide bonds (Pedersen et al., Mol Cell Biol 29, 3319-31 (2009)). Anexemplary sequence of human p95HER2 is given in SEQ ID NO: 145.

“HER3” or “ErbB3” (human epidermal growth factor receptor 3), like theother members of the ErbB receptor tyrosine kinase family, consists ofan extracellular domain, a transmembrane domain, and an intracellulardomain. The extracellular domain contains four subdomains (I-IV).Subdomains I and III are leucine-rich and are primarily involved inligand binding. Subdomains II and IV are cysteine-rich and most likelycontribute to protein conformation and stability through the formationof disulfide bonds. Subdomain II also contains the dimerization looprequired for dimer formation. The cytoplasmic domain contains ajuxtamembrane segment, a kinase domain, and a C-terminal domain. Whileno evidence has been found that ErbB3 overexpression, constitutiveactivation, or mutation alone is oncogenic,en.wikipedia.org/wiki/ERBB3-cite_note-pmid8632008-18 the protein as aheterodimerization partner, most critically with ErbB2, is implicated ingrowth, proliferation, chemotherapeutic resistance, and the promotion ofinvasion and metastasis. ErbB3 is associated with targeted therapeuticresistance in numerous cancers. The amino acid sequence of human HER3 isshown in UniProt accession no. P21860 (version 224, SEQ ID NO:471)

A “B cell surface antigen” as used herein refers to an antigenicdeterminant presented on the surface of a B lymphocyte, particularly amalignant B lymphocyte (in that case the antigen also being referred toas “malignant B-cell surface antigen”). Several B-cell surface antigensare interesting in terms of immunotherapy of hematologic malignantneoplasms. In one aspect, the B cell surface antigen is selected fromthe group consisting of CD19, CD79b, CD20, CD22 and CD37.

The term “CD19” refers to B-lymphocyte antigen CD19, also known asB-lymphocyte surface antigen B4 or T-cell surface antigen Leu-12 andincludes any native CD19 from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), unless otherwise indicated.The amino acid sequence of human CD19 is shown in Uniprot accession no.P15391 (version 160, SEQ ID NO:434). The term encompasses “full-length”unprocessed human CD19 as well as any form of human CD19 that resultsfrom processing in the cell as long as the antibody as reported hereinbinds thereto. CD19 is a structurally distinct cell surface receptorexpressed on the surface of human B cells, including, but not limitedto, pre-B cells, B cells in early development {i.e., immature B cells),mature B cells through terminal differentiation into plasma cells, andmalignant B cells. CD19 is expressed by most pre-B acute lymphoblasticleukemias (ALL), non-Hodgkin's lymphomas, B cell chronic lymphocyticleukemias (CLL), pro-lymphocytic leukemias, hairy cell leukemias, commonacute lymphocytic leukemias, and some Null-acute lymphoblasticleukemias. The expression of CD19 on plasma cells further suggests itmay be expressed on differentiated B cell tumors such as multiplemyeloma. Therefore, the CD19 antigen is a target for immunotherapy inthe treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemiaand/or acute lymphoblastic leukemia.

“CD79b” refers to B-cell antigen receptor complex-associated proteinbeta chain, also known as Ig-beta or B cell specific glycoprotein B29,and includes any native CD79b from any vertebrate source, includingmammals such as primates (e.g. humans) non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The amino acid sequence of human CD79b is shown in Uniprotaccession no. P40259 (version 180, SEQ ID NO:435). CD79b is a 39 KDaprotein exclusively expressed on B cells and, in cooperation with CD79a,initiates the signal transduction cascade downstream of the BCR, whichleads to internalization of the BCR complex, its translocation to theendosomes, and antigen presentation. CD79 (composed of subunits CD79aand CD79b) is a heterodimeric signal-transduction component of theB-cell receptor, ubiquitously expressed in mature B-cell lymphomas andplaced on the cell surface by the earliest committed B-cell progenitorsbefore expression of immunoglobulin μ. The term “CD79b” encompasses“full-length,” unprocessed CD79b as well as any form of CD79b thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of CD79b, e.g., splice variants or allelic variants.

“CD20” refers to B-lymphocyte antigen CD20, also known as B-lymphocytesurface antigen B1 or Leukocyte surface antigen Leu-16, and includes anynative CD20 from any vertebrate source, including mammals such asprimates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The amino acidsequence of human CD20 is shown in Uniprot accession no. P11836 (version149, SEQ ID NO:436). CD20 is a hydrophobic transmembrane protein with amolecular weight of approximately 35 kD expressed on pre-B and mature Blymphocytes. The corresponding human gene is membrane-spanning4-domains, subfamily A, member 1, also known as MS4A1. This gene encodesa member of the membrane-spanning 4A gene family. Members of thisnascent protein family are characterized by common structural featuresand similar intron/exon splice boundaries and display unique expressionpatterns among hematopoietic cells and nonlymphoid tissues. This geneencodes the B-lymphocyte surface molecule which plays a role in thedevelopment and differentiation of B-cells into plasma cells. Thisfamily member is localized to 11q12, among a cluster of family members.Alternative splicing of this gene results in two transcript variantswhich encode the same protein. The term “CD20” encompasses“full-length,” unprocessed CD20 as well as any form of CD20 that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of CD20, e.g., splice variants or allelic variants.

“CD22” refers to B-cell receptor CD22, also known as B-lymphocyte celladhesion molecule or SIGLEC2, and includes any native CD22 from anyvertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The amino acid sequence of human CD22is shown in Uniprot accession no. P20273 (version 209, SEQ ID NO:437).CD22 is a molecule belonging to the SIGLEC family of lectins and isfound on the surface of mature B cells and to lesser extent on someimmature B cells. CD22 is thus a B cell restricted cell surfacephosphoglycoprotein of 130-150 kDa and is capable of modulating Blymphocyte antigen receptor (BCR)-mediated signals, as well as thegeneration of BCR-independent signals. The term “CD22” encompasses“full-length,” unprocessed CD22 as well as any form of CD22 that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of CD22, e.g., splice variants or allelic variants.

“CD37” refers to Leukocyte antigen CD37, also known as Tetraspanin-26(Tspan-26), and includes any native CD37 from any vertebrate source,including mammals such as primates (e.g. humans) non-human primates(e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unlessotherwise indicated. The amino acid sequence of human CD37 is shown inUniprot accession no. P11049 (version 162, SEQ ID NO:438). CD37expression is restricted to cells of the immune system, with highestabundance on mature B cells, and lower expression is found on T cellsand myeloid cells. The glycoprotein CD37 is a member of thetransmembrane 4 superfamily and controls both humoral and cellularimmune responses. The term “CD37” encompasses “full-length,” unprocessedCD37 as well as any form of CD37 that results from processing in thecell. The term also encompasses naturally occurring variants of CD37,e.g., splice variants or allelic variants.

A “Multiple Myeloma (MM) cell surface antigen” as used herein refers toan antigenic determinant presented on the surface of a Multiple Myeloma(MM) cell. Several MM cell surface antigens are interesting in terms ofimmunotherapy of Multiple Myeloma. In one aspect, the MM cell surfaceantigen is selected from the group consisting of CD38, BCMA and GPRC5D.

The term “CD38”, also known as cluster of differentiation 38 or cyclicADP ribose hydrolase, is a glycoprotein found on the surface of manyimmune cells (white blood cells), including CD4+, CD8+, B lymphocytesand natural killer cells. CD38 also functions in cell adhesion, signaltransduction and calcium signaling. Under normal conditions, CD38 isexpressed at relatively low levels on myeloid and lymphoid cells and insome non-hematopoietic tissues. In contrast, normal plasma cells andmultiple myeloma (MM) cells have high levels of CD38 expression, whichmakes CD38 an interesting target for targeting cell surface molecules inMM. CD38 as used herein refers to any CD38 protein from any vertebratesource, including mammals such as primates (e.g. humans) non-humanprimates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats),unless otherwise indicated. The amino acid sequence of human CD38 isshown in UniProt (world wide web.uniprot.org) accession no. P28907 (SEQID NO:474).

The term “BCMA” refers to B cell maturation antigen, also termed tumornecrosis factor receptor superfamily member 17 (TNFRS17), and is a typeIII transmembrane protein without a signal-peptide and containingcysteine-rich extracellular domains. BCMA is expressed at significantlyhigher levels in all patient MM cells but not on other normal tissuesexcept normal plasma cells. BCMA, along with two related TNFRsuperfamily B-cell activation factor receptor (BAFF-R) and transmembraneactivator and calcium modulator and cyclophilin ligand interactor(TACI), critically regulate B cell proliferation and survival, as wellas maturation and differentiation into plasma cells. These threefunctionally related receptors support long-term survival of B cells atdifferent stages of development by binding to BAFF and/or APRIL, theircognate ligands. BCMA as used herein refers to any BCMA protein from anyvertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The amino acid sequence of human BCMAis shown in UniProt (world wide web.uniprot.org) accession no. Q02223(SEQ ID NO:475).

The term “GPRC5D” refers to G protein-coupled receptor class C group 5member D, a target identified from plasma cells in multiple myelomausing RNA-sequencing. It has been reported that GPRC5D is associatedwith poor prognosis and tumour load in multiple myeloma patients. GPRC5Drefers to any GPRC5D protein from any vertebrate source, includingmammals such as primates (e.g. humans) non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The amino acid sequence of human GPRC5D is shown in UniProt(world wide web.uniprot.org) accession no. Q9NZD1 (SEQ ID NO:476).

The term “CD28” (Cluster of differentiation 28, Tp44) refers to any CD28protein from any vertebrate source, including mammals such as primates(e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents(e.g. mice and rats), unless otherwise indicated. CD28 is expressed on Tcells and provides co-stimulatory signals required for T cell activationand survival. T cell stimulation through CD28 in addition to the T-cellreceptor (TCR) can provide a potent signal for the production of variousinterleukins. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2)proteins and is the only B7 receptor constitutively expressed on naive Tcells. The amino acid sequence of human CD28 is shown in UniProt (worldwide web.uniprot.org) accession no. P10747 (SEQ ID NO:1).

An “agonistic antibody” refers to an antibody that comprises anagonistic function against a given receptor. In general, when an agonistligand (factor) binds to a receptor, the tertiary structure of thereceptor protein changes, and the receptor is activated (when thereceptor is a membrane protein, a cell growth signal or such is usuallytransducted). If the receptor is a dimer-forming type, an agonisticantibody can dimerize the receptor at an appropriate distance and angle,thus acting similarly to a ligand. An appropriate anti-receptor antibodycan mimic dimerization of receptors performed by ligands, and thus canbecome an agonistic antibody.

A “CD28 agonistic antigen binding molecule” or “CD28 conventionalagonistic antigen binding molecule” is an antigen binding molecule thatmimicks CD28 natural ligands (CD80 or CD86) in their role to enhance Tcell activation in presence of a T cell receptor signal (“signal 2”). AT cell needs two signals to become fully activated. Under physiologicalconditions “signal 1” arises form the interaction of T cell receptor(TCR) molecules with peptide/major histocompatibility complex (MEW)complexes on antigen presenting cells (APCs) and “signal 2” is providedby engagement of a costimulatory receptor, e.g. CD28. A CD28 agonisticantigen binding molecule is able to costimulate T cells (signal 2). Itis also able to induce T cell proliferation and cytokine secretion incombination with a molecule with specificity for the TCR complex,however the CD28 agonistic antigen binding molecule is not capable offully activating T cells without additional stimulation of the TCR.There is however a subclass of CD28 specific antigen binding molecules,the so-called CD28 superagonistic antigen binding molecules. A “CD28superagonistic antigen binding molecule” is a CD28 antigen bindingmolecule which is capable of fully activating I cells without additionalstimulation of the TCR. A CD28 superagonistic antigen binding moleculeis capable to induce T cell proliferation and cytokine secretion withoutprior T cell activation (signal 1).

The term “variable domain” or “variable region” refers to the domain ofan antibody heavy or light chain that is involved in binding the antigenbinding molecule to antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). See,e.g., Kindt et al., Kuby Immunology, 6th ed., W. H. Freeman and Co.,page 91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antigen binding variable domain which arehypervariable in sequence and which determine antigen bindingspecificity, for example “complementarity determining regions” (“CDRs”).Generally, antigen binding domains comprise six CDRs: three in the VH(CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3).Exemplary CDRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, MD (1991)); and    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)).

Unless otherwise indicated, the CDRs are determined according to Kabatet al., supra. One of skill in the art will understand that the CDRdesignations can also be determined according to Chothia, supra,McCallum, supra, or any other scientifically accepted nomenclature.Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

As used herein, the term “affinity matured” in the context of antigenbinding molecules (e.g., antibodies) refers to an antigen bindingmolecule that is derived from a reference antigen binding molecule,e.g., by mutation, binds to the same antigen, preferably binds to thesame epitope, as the reference antibody; and has a higher affinity forthe antigen than that of the reference antigen binding molecule.Affinity maturation generally involves modification of one or more aminoacid residues in one or more CDRs of the antigen binding molecule.Typically, the affinity matured antigen binding molecule binds to thesame epitope as the initial reference antigen binding molecule.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g. IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “CH1 domain” denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 118 to EUposition 215 (EU numbering system according to Kabat). In one aspect, aCH1 domain has the amino acid sequence of ASTKGPSVFP LAPSSKSTSGGTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKKV(SEQ ID NO: 477). Usually, a segment having the aminoacid sequence of EPKSC (SEQ ID NO:480) is following to link the CH1domain to the hinge region,

The term “hinge region” denotes the part of an antibody heavy chainpolypeptide that joins in a wild-type antibody heavy chain the CH1domain and the CH2 domain, e.g. from about position 216 to aboutposition 230 according to the EU number system of Kabat, or from aboutposition 226 to about position 230 according to the EU number system ofKabat. The hinge regions of other IgG subclasses can be determined byaligning with the hinge-region cysteine residues of the IgG1 subclasssequence. The hinge region is normally a dimeric molecule consisting oftwo polypeptides with identical amino acid sequence. The hinge regiongenerally comprises up to 25 amino acid residues and is flexibleallowing the associated target binding sites to move independently. Thehinge region can be subdivided into three domains: the upper, themiddle, and the lower hinge domain (see e.g. Roux, et al., J. Immunol.161 (1998) 4083).

In one aspect, the hinge region has the amino acid sequence DKTHTCPXCP(SEQ ID NO: 481), wherein X is either S or P. In one aspect, the hingeregion has the amino acid sequence HTCPXCP (SEQ ID NO: 482), wherein Xis either S or P. In one aspect, the hinge region has the amino acidsequence CPXCP (SEQ ID NO:483), wherein X is either S or P.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. An IgG Fc region comprises an IgG CH2and an IgG CH3 domain.

The “CH2 domain” of a human IgG Fc region usually extends from an aminoacid residue at about EU position 231 to an amino acid residue at aboutEU position 340 (EU numbering system according to Kabat). In one aspect,a CH2 domain has the amino acid sequence of APELLGGPSV FLFPPKPKDTLMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 478). The CH2 domain is uniquein that it is not closely paired with another domain. Rather, twoN-linked branched carbohydrate chains are interposed between the two CH2domains of an intact native Fc-region. It has been speculated that thecarbohydrate may provide a substitute for the domain-domain pairing andhelp stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206.In one embodiment, a carbohydrate chain is attached to the CH2 domain.The CH2 domain herein may be a native sequence CH2 domain or variant CH2domain.

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region denotes the part of an antibody heavy chainpolypeptide that extends approximately from EU position 341 to EUposition 446 (EU numbering system according to Kabat). In one aspect,the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 479). The CH3 region herein maybe a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3domain with an introduced “protuberance” (“knob”) in one chain thereofand a corresponding introduced “cavity” (“hole”) in the other chainthereof; see U.S. Pat. No. 5,821,333, expressly incorporated herein byreference). Such variant CH3 domains may be used to promoteheterodimerization of two non-identical antibody heavy chains as hereindescribed. In one embodiment, a human IgG heavy chain Fc region extendsfrom Cys226, or from Pro230, to the carboxyl-terminus of the heavychain. However, the C-terminal lysine (Lys447) of the Fc region may ormay not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, M D, 1991.

The “knob-into-hole” technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc region, thus furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term “wild-type Fc domain” denotes an amino acid sequence identicalto the amino acid sequence of an Fc domain found in nature. Wild-typehuman Fc domains include a native human IgG1 Fc-region (non-A and Aallotypes), native human IgG2 Fc-region, native human IgG3 Fc-region,and native human IgG4 Fc-region as well as naturally occurring variantsthereof. Wild-type Fc-regions are denoted in SEQ ID NO: 484 (IgG1,caucasian allotype), SEQ ID NO: 485 (IgG1, afroamerican allotype), SEQID NO: 486 (IgG2), SEQ ID NO: 487 (IgG3) and SEQ ID NO:488 (IgG4).

The term “variant (human) Fc domain” denotes an amino acid sequencewhich differs from that of a “wild-type” (human) Fc domain amino acidsequence by virtue of at least one “amino acid mutation”. In one aspect,the variant Fc-region has at least one amino acid mutation compared to anative Fc-region, e.g. from about one to about ten amino acid mutations,and in one aspect from about one to about five amino acid mutations in anative Fc-region. In one aspect, the (variant) Fc-region has at leastabout 95% homology with a wild-type Fc-region.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

Fc receptor binding dependent effector functions can be mediated by theinteraction of the Fc-region of an antibody with Fc receptors (FcRs),which are specialized cell surface receptors on hematopoietic cells. Fcreceptors belong to the immunoglobulin superfamily, and have been shownto mediate both the removal of antibody-coated pathogens by phagocytosisof immune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody, via antibody dependent cell mediated cytotoxicity (ADCC) (seee.g. Van de Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49(1991) 511-524). FcRs are defined by their specificity forimmunoglobulin isotypes: Fc receptors for IgG antibodies are referred toas FcγR. Fc receptor binding is described e.g. in Ravetch, J. V. andKinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., etal., Immunomethods 4 (1994) de Haas, M., et al., J. Lab. Clin. Med. 126(1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998)231-248.

Cross-linking of receptors for the Fc-region of IgG antibodies (FcγR)triggers a wide variety of effector functions including phagocytosis,antibody-dependent cellular cytotoxicity, and release of inflammatorymediators, as well as immune complex clearance and regulation ofantibody production. In humans, three classes of FcγR have beencharacterized, which are:

-   -   FcγRI (CD64) binds monomeric IgG with high affinity and is        expressed on macrophages, monocytes, neutrophils and        eosinophils. Modification in the Fc-region IgG at least at one        of the amino acid residues E233-G236, P238, D265, N297, A327 and        P329 (numbering according to EU index of Kabat) reduce binding        to FcγRI. IgG2 residues at positions 233-236, substituted into        IgG1 and IgG4, reduced binding to FcγRI by 10³-fold and        eliminated the human monocyte response to antibody-sensitized        red blood cells (Armour, K. L., et al., Eur. J. Immunol.        29 (1999) 2613-2624).    -   FcγRII (CD32) binds complexed IgG with medium to low affinity        and is widely expressed. This receptor can be divided into two        sub-types, FcγRIIA and FcγRIM FcγRIIA is found on many cells        involved in killing (e.g. macrophages, monocytes, neutrophils)        and seems able to activate the killing process. FcγRIIB seems to        play a role in inhibitory processes and is found on B cells,        macrophages and on mast cells and eosinophils. On B-cells it        seems to function to suppress further immunoglobulin production        and isotype switching to, for example, the IgE class. On        macrophages, FcγRIIB acts to inhibit phagocytosis as mediated        through FcγRIIA. On eosinophils and mast cells the B-form may        help to suppress activation of these cells through IgE binding        to its separate receptor. Reduced binding for FcγRIIA is found        e.g. for antibodies comprising an IgG Fc-region with mutations        at least at one of the amino acid residues E233-G236, P238,        D265, N297, A327, P329, D270, Q295, A327, R292, and K414        (numbering according to EU index of Kabat).    -   FcγRIII (CD16) binds IgG with medium to low affinity and exists        as two types. FcγRIIIA is found on NK cells, macrophages,        eosinophils and some monocytes and T cells and mediates ADCC.        FcγRIIIB is highly expressed on neutrophils. Reduced binding to        FcγRIIIA is found e.g. for antibodies comprising an IgG        Fc-region with mutation at least at one of the amino acid        residues E233-G236, P238, D265, N297, A327, P329, D270, Q295,        A327, 5239, E269, E293, Y296, V303, A327, K338 and D376        (numbering according to EU index of Kabat).

Mapping of the binding sites on human IgG1 for Fc receptors, the abovementioned mutation sites and methods for measuring binding to FcγRI andFcγRIIA are described in Shields, R. L., et al. J. Biol. Chem. 276(2001) 6591-6604.

The term “ADCC” or “antibody-dependent cellular cytotoxicity” is animmune mechanism leading to lysis of antibody-coated target cells byimmune effector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example, the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).For example, the capacity of the antibody to induce the initial stepsmediating ADCC is investigated by measuring their binding to Fcγreceptors expressing cells, such as cells, recombinantly expressingFcγRI and/or FcγRIIA or NK cells (expressing essentially FcγRIIIA) Inparticular, binding to FcγR on NK cells is measured.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcγRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637, version 141).

An “ectodomain” is the domain of a membrane protein that extends intothe extracellular space (i.e. the space outside the target cell).Ectodomains are usually the parts of proteins that initiate contact withsurfaces, which leads to signal transduction.

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2 to 20 amino acids. Peptide linkers areknown in the art or are described herein. Suitable, non-immunogeniclinker peptides are, for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n)peptide linkers, wherein “n” is generally a number between 1 and 5,typically between 2 and 4, in particular 2, i.e. the peptides selectedfrom the group consisting of GGGGS (SEQ ID NO:146) GGGGSGGGGS (SEQ IDNO:147), SGGGGSGGGG (SEQ ID NO:148) and GGGGSGGGGSGGGG (SEQ ID NO:149),but also include the sequences GSPGSSSSGS (SEQ ID NO:150), (G₄S) 3 (SEQID NO:151), (G₄S)₄ (SEQ ID NO:152), GSGSGSGS (SEQ ID NO:153), GSGSGNGS(SEQ ID NO:154), GGSGSGSG (SEQ ID NO:155), GGSGSG (SEQ ID NO:156), GGSG(SEQ ID NO:157), GGSGNGSG (SEQ ID NO:158), GGNGSGSG (SEQ ID NO:159) andGGNGSG (SEQ ID NO:160). Peptide linkers of particular interest are (G₄S)(SEQ ID NO:146), (G₄S)₂ or GGGGSGGGGS (SEQ ID NO:147), (G₄S)₃ (SEQ IDNO:151) and (G₄S)₄ (SEQ ID NO:152).

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

By “fused” or “connected” is meant that the components (e.g. apolypeptide and an ectodomain of said TNF ligand family member) arelinked by peptide bonds, either directly or via one or more peptidelinkers.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide (protein) sequence is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN. SAWIor Megalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, California, or may be compiled from thesource code. The ALIGN-2 program should be compiled for use on a UNIXoperating system, including digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary. In situationswhere ALIGN-2 is employed for amino acid sequence comparisons, the %amino acid sequence identity of a given amino acid sequence A to, with,or against a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows: 100 times the fraction X/Y, whereX is the number of amino acid residues scored as identical matches bythe sequence alignment program ALIGN-2 in that program's alignment of Aand B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In certain embodiments, amino acid sequence variants of the CD28 antigenbinding molecules provided herein are contemplated. For example, it maybe desirable to improve the binding affinity and/or other biologicalproperties of the CD28 antigen binding molecules. Amino acid sequencevariants of the CD28 antigen binding molecules may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the molecules, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.Sites of interest for substitutional mutagenesis include the HVRs andFramework (FRs). Conservative substitutions are provided in Table Bunder the heading “Preferred Substitutions” and further described belowin reference to amino acid side chain classes (1) to (6). Amino acidsubstitutions may be introduced into the molecule of interest and theproducts screened for a desired activity, e.g., retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE A Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “amino acid sequence variants” includes substantial variantswherein there are amino acid substitutions in one or more hypervariableregion residues of a parent antigen binding molecule (e.g. a humanizedor human antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antigen binding molecule and/or will havesubstantially retained certain biological properties of the parentantigen binding molecule. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antigen binding molecules displayed on phage and screened for aparticular biological activity (e.g. binding affinity). In certainembodiments, substitutions, insertions, or deletions may occur withinone or more HVRs so long as such alterations do not substantially reducethe ability of the antigen binding molecule to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. A useful method for identification of residues orregions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells(1989) Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys, andGlu) are identified and replaced by a neutral or negatively chargedamino acid (e.g., alanine or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of insertions include CD28antigen binding molecules with a fusion to the N- or C-terminus to apolypeptide which increases the serum half-life of the CD28 antigenbinding molecules.

In certain embodiments, the CD28 antigen binding molecules providedherein are altered to increase or decrease the extent to which theantibody is glycosylated. Glycosylation variants of the molecules may beconveniently obtained by altering the amino acid sequence such that oneor more glycosylation sites is created or removed. Where the agonisticICOS-binding molecule comprises an Fc domain, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in agonistic ICOS-binding molecules may be made in orderto create variants with certain improved properties. In one aspect,variants of agonistic ICOS-binding molecules are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. Such fucosylation variants may haveimproved ADCC function, see e.g. US Patent Publication Nos. US2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co.,Ltd). Further variants of the CD28 antigen binding molecules of theinvention include those with bisected oligosaccharides, e.g., in which abiantennary oligosaccharide attached to the Fc region is bisected byGlcNAc. Such variants may have reduced fucosylation and/or improved ADCCfunction, see for example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).Variants with at least one galactose residue in the oligosaccharideattached to the Fc region are also provided. Such antibody variants mayhave improved CDC function and are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, it may be desirable to create cysteineengineered variants of the CD28 antigen binding molecules of theinvention, e.g., “thioMAbs,” in which one or more residues of themolecule are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of themolecule. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate. Incertain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and 5400 (EU numbering) of theheavy chain Fc region. Cysteine engineered antigen binding molecules maybe generated as described, e.g., in U.S. Pat. No. 7,521,541.

In certain aspects, the CD28 antigen binding molecules provided hereinmay be further modified to contain additional non-proteinaceous moietiesthat are known in the art and readily available. The moieties suitablefor derivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether thebispecific antibody derivative will be used in a therapy under definedconditions, etc. In another aspect, conjugates of an antibody andnon-proteinaceous moiety that may be selectively heated by exposure toradiation are provided. In one embodiment, the non-proteinaceous moietyis a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102(2005) 11600-11605). The radiation may be of any wavelength, andincludes, but is not limited to, wavelengths that do not harm ordinarycells, but which heat the non-proteinaceous moiety to a temperature atwhich cells proximal to the antibody-non-proteinaceous moiety arekilled. In another aspect, immunoconjugates of the CD28 antigen bindingmolecules provided herein may be obtained. An “immunoconjugate” is anantibody conjugated to one or more heterologous molecule(s), includingbut not limited to a cytotoxic agent.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide. Each nucleotide is composed of a base,specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine(G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyriboseor ribose), and a phosphate group. Often, the nucleic acid molecule isdescribed by the sequence of bases, whereby said bases represent theprimary structure (linear structure) of a nucleic acid molecule. Thesequence of bases is typically represented from 5′ to 3′. Herein, theterm nucleic acid molecule encompasses deoxyribonucleic acid (DNA)including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleicacid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNAor RNA, and mixed polymers comprising two or more of these molecules.The nucleic acid molecule may be linear or circular. In addition, theterm nucleic acid molecule includes both, sense and antisense strands,as well as single stranded and double stranded forms. Moreover, theherein described nucleic acid molecule can contain naturally occurringor non-naturally occurring nucleotides. Examples of non-naturallyoccurring nucleotides include modified nucleotide bases with derivatizedsugars or phosphate backbone linkages or chemically modified residues.Nucleic acid molecules also encompass DNA and RNA molecules which aresuitable as a vector for direct expression of an antibody of theinvention in vitro and/or in vivo, e.g., in a host or patient. Such DNA(e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified.For example, mRNA can be chemically modified to enhance the stability ofthe RNA vector and/or expression of the encoded molecule so that mRNAcan be injected into a subject to generate the antibody in vivo (seee.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2 101 823B1).

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable excipient includes,but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the moleculesof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “combination treatment” or “co-administration” as noted hereinencompasses combined administration (where two or more therapeuticagents are included in the same or separate formulations), and separateadministration, in which case, administration of an antibody as reportedherein can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent or agents, preferablyan antibody or antibodies.

By “B cell proliferative disorder” is meant a disease wherein the numberof B cells in a patient is increased as compared to the number of Bcells in a healthy subject, and particularly wherein the increase in thenumber of B cells is the cause or hallmark of the disease.

The term “hematological cancer” refers to or describes the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth/proliferation. Thus, the term cancer as used herein refers toproliferative diseases, such as carcinoma, lymphomas (e.g., Hodgkin'sand non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Inparticular, the term cancer refers to a B-cell proliferative disorder.In one aspect, the cancer is selected from the group consisting ofNon-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zonelymphoma (MZL), Multiple myeloma (MM), and Hodgkin lymphoma (HL).

The term “cancer” refers to or describes the physiological condition inmammals that is typically characterized by unregulated cellgrowth/proliferation. Thus, the term cancer as used herein refers toproliferative diseases, such as carcinoma, lymphomas (e.g., Hodgkin'sand non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Inparticular, the term cancer includes lymphocytic leukemias, lung cancer,non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer,bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, gastriccancer, colon cancer, breast cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, cancer of thebladder, cancer of the kidney or ureter, renal cell carcinoma, carcinomaof the renal pelvis, mesothelioma, hepatocellular cancer, biliarycancer, neoplasms of the central nervous system (CNS), spinal axistumors, brain stem glioma, glioblastoma multiforme, astrocytomas,schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cellcarcinomas, pituitary adenoma and Ewings sarcoma, including refractoryversions of any of the above cancers, or a combination of one or more ofthe above cancers. In one aspect, the cancer is a solid tumor. Inanother aspect, the cancer is a haematological cancer, particularlyleukemia, most particularly acute lymphoblastic leukemia (ALL) or acutemyelogenous leukemia (AML).

Bispecific Agonistic CD28 Antigen Binding Molecules of the Invention

The invention provides novel bispecific agonistic CD28 antigen bindingmolecules with particularly advantageous properties such asproducibility, stability, binding affinity, biological activity,targeting efficiency, reduced toxicity, an extended dosage range thatcan be given to a patient and thereby a possibly enhanced efficacy. Thenovel bispecific agonistic CD28 antigen binding molecules comprise an Fcdomain composed of a first and a second subunit capable of stableassociation comprising one or more amino acid substitution that reducesthe binding affinity of the antigen binding molecule to an Fc receptorand/or effector function (Fc silent) and thus unspecific cross-linkingvia Fc receptors is avoided. Instead, they comprise at least one antigenbinding domain capable of specific binding to a tumor-associated antigensuch as Fibroblast Activation Protein (FAP) or Carcinoembryonic Antigen(CEA) which causes cross-linking at the tumor site. Thus, tumor-specificT cell activation is achieved.

Herein provided is a bispecific agonistic CD28 antigen binding moleculewith monovalent binding to CD28, comprising

-   -   (a) one antigen binding domains capable of specific binding to        CD28,    -   (b) at least one antigen binding domain capable of specific        binding to a tumor-associated antigen, and    -   (c) an Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, a bispecific agonistic CD28 antigen binding molecule asdefined herein before is provided, wherein the Fc domain is an IgG,particularly an IgG1 Fc domain or an IgG4 Fc domain. In one particularaspect, the Fc domain composed of a first and a second subunit capableof stable association is an IgG1 Fc domain. The Fc domain comprises oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or reduces or abolisheseffector function. In one aspect, the Fc domain comprises the amino acidsubstitutions L234A and L235A (numbering according to Kabat EU index).In one aspect, the Fc domain is of human IgG1 subclass and comprises theamino acid mutations L234A, L235A and P329G (numbering according toKabat EU index). In one aspect, a bispecific agonistic CD28 antigenbinding molecule is provided, wherein the antigen binding moleculecomprises an Fc domain composed of a first and a second subunit capableof stable association, wherein the first subunit comprises the aminoacid sequence of SEQ ID NO:176 and the second subunit comprise the aminoacid sequence of SEQ ID NO:177.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as defined herein before, wherein the antigen binding domaincapable of specific binding to CD28 comprises

-   -   (i) a heavy chain variable region (V_(H)CD28) comprising a heavy        chain complementary determining region CDR-H1 of SEQ ID NO: 20,        a CDR-H2 of SEQ ID NO: 21, and a CDR-H3 of SEQ ID NO: 22, and a        light chain variable region (V_(L)CD28) comprising a light chain        complementary determining region CDR-L1 of SEQ ID NO: 23, a        CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQ ID NO: 25; or    -   (ii) a heavy chain variable region (V_(H)CD28) comprising a        CDR-H1 of SEQ ID NO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3        of SEQ ID NO: 38, and a light chain variable region (V_(L)CD28)        comprising a CDR-L1 of SEQ ID NO: 39, a CDR-L2 of SEQ ID NO: 40        and a CDR-L3 of SEQ ID NO: 41.

In one aspect, the antigen binding domain capable of specific binding toCD28 of the bispecific agonistic CD28 antigen binding molecule comprisesa heavy chain variable region (V_(H)CD28) comprising a CDR-H1 of SEQ IDNO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3 of SEQ ID NO: 38, and alight chain variable region (V_(L)CD28) comprising a CDR-L1 of SEQ IDNO: 39, a CDR-L2 of SEQ ID NO: 40 and a CDR-L3 of SEQ ID NO: 41.

In another aspect, the antigen binding domain capable of specificbinding to CD28 of the bispecific agonistic CD28 antigen bindingmolecule comprises a heavy chain variable region (V_(H)CD28) comprisinga CDR-H1 of SEQ ID NO: 20, a CDR-H2 of SEQ ID NO: 21, and a CDR-H3 ofSEQ ID NO: 22, and a light chain variable region (V_(L)CD28) comprisinga CDR-L1 of SEQ ID NO: 23, a CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQID NO: 25.

Furthermore, provided is a bispecific agonistic CD28 antigen bindingmolecule as defined herein before, wherein the antigen binding domaincapable of specific binding to CD28 comprises a heavy chain variableregion (V_(H)CD28) comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:26, and a light chain variable region (V_(L)CD28)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:27.

In another aspect, provided is bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD28 comprises

-   -   (a) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:47 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:54, or    -   (b) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:47 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:27, or    -   (c) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:51 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:61, or    -   (d) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:46 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:53, or    -   (e) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:46 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:54, or    -   (f) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:46 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:59, or    -   (g) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:46 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:27, or    -   (h) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:43 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:27, or    -   (i) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:42 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:53, or    -   (j) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:42 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:59, or    -   (k) the CDRs of the heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:42 and the CDRs        of the light chain variable region (V_(L)CD28) comprising the        amino acid sequence of SEQ ID NO:27.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD28 comprises the CDRs of the heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:47 and theCDRs of the light chain variable region (V_(L)CD28) comprising the aminoacid sequence of SEQ ID NO:54. In another aspect, the antigen bindingdomain capable of specific binding to CD28 of the bispecific agonisticCD28 antigen binding molecule comprises a heavy chain variable region(V_(H)CD28) comprising a CDR-H1 of SEQ ID NO: 489, a CDR-H2 of SEQ IDNO: 490, and a CDR-H3 of SEQ ID NO: 491, and a light chain variableregion (V_(L)CD28) comprising a CDR-L1 of SEQ ID NO: 492, a CDR-L2 ofSEQ ID NO: 493 and a CDR-L3 of SEQ ID NO: 494.

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule, wherein the antigen binding domain capable of specificbinding to CD28 comprises the CDRs of the heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:46 and theCDRs of the light chain variable region (V_(L)CD28) comprising the aminoacid sequence of SEQ ID NO:53. In another aspect, the antigen bindingdomain capable of specific binding to CD28 of the bispecific agonisticCD28 antigen binding molecule comprises a heavy chain variable region(V_(H)CD28) comprising a CDR-H1 of SEQ ID NO: 495, a CDR-H2 of SEQ IDNO: 496, and a CDR-H3 of SEQ ID NO: 497, and a light chain variableregion (V_(L)CD28) comprising a CDR-L1 of SEQ ID NO: 498, a CDR-L2 ofSEQ ID NO: 499 and a CDR-L3 of SEQ ID NO: 500.

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule, wherein the antigen binding domain capable of specificbinding to CD28 comprises the CDRs of the heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:42 and theCDRs of the light chain variable region (V_(L)CD28) comprising the aminoacid sequence of SEQ ID NO:27. In another aspect, the antigen bindingdomain capable of specific binding to CD28 of the bispecific agonisticCD28 antigen binding molecule comprises a heavy chain variable region(V_(H)CD28) comprising a CDR-H1 of SEQ ID NO: 501, a CDR-H2 of SEQ IDNO: 502, and a CDR-H3 of SEQ ID NO: 503, and a light chain variableregion (V_(L)CD28) comprising a CDR-L1 of SEQ ID NO: 504, a CDR-L2 ofSEQ ID NO: 505 and a CDR-L3 of SEQ ID NO: 506.

In a further aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 andSEQ ID NO:51, and a light chain variable region (V_(L)CD28) comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:27, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ IDNO:61.

In another aspect, provided is bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD28 comprises

-   -   (a) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (b) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (c) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:51 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:61, or    -   (d) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (e) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (f) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (g) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (h) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:43 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (j) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (k) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27.

In one aspect, a bispecific agonistic CD28 antigen binding molecule isprovided, wherein the antigen binding domain capable of specific bindingto CD28 binds to CD28 with an reduced affinity compared to an antigenbinding domain comprising a heavy chain variable region (V_(H)CD28)comprising the amino acid sequence of SEQ ID NO:26 and a light chainvariable region (V_(L)CD28) comprising the amino acid sequence of SEQ IDNO:27. The affinity is measured by flow cytometry as binding to CHOcells expressing CD28. In one aspect, the antigen binding domain capableof specific binding to CD28 binds to CD28 with an reduced affinitycompared to an antigen binding domain comprising a heavy chain variableregion (V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:26and a light chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:27 comprises the CDR-H1, CDR-H2 and CDR-H3 of theheavy chain variable region (V_(H)CD28) comprising the amino acidsequence of SEQ ID NO:47 and the CDR-L1, CDR-L2 and CDR-L3 of the lightchain variable region (V_(L)CD28) comprising the amino acid sequence ofSEQ ID NO:54. In one aspect, the antigen binding domain capable ofspecific binding to CD28 with reduced affinity compared to an antigenbinding domain comprising a heavy chain variable region (V_(H)CD28)comprising the amino acid sequence of SEQ ID NO:26 and a light chainvariable region (V_(L)CD28) comprising the amino acid sequence of SEQ IDNO:27 comprises a heavy chain variable region (V_(H)CD28) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:47, and a lightchain variable region (V_(L)CD28) comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:54.

In one particular aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:47 and alight chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:54.

In another particular aspect, a bispecific agonistic CD28 antigenbinding molecule is provided, wherein the antigen binding domain capableof specific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:46 and alight chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:53.

In further particular aspect, a bispecific agonistic CD28 antigenbinding molecule is provided, wherein the antigen binding domain capableof specific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:42 and alight chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:27.

CEA-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

In one aspect, a bispecific agonistic CD28 antigen binding molecule isprovided, wherein the antigen binding domain capable of specific bindingto a tumor-associated antigen is an antigen binding domain capable ofspecific binding to Carcinoembryonic Antigen (CEA).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to CEA comprises

-   -   (i) a heavy chain variable region (V_(H)CEA) comprising a CDR-H1        comprising the amino acid sequence of SEQ ID NO:188, a CDR-H2        comprising the amino acid sequence of SEQ ID NO:189, and a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:190, and        a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:191, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:192, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:193; or    -   (ii) a heavy chain variable region (V_(H)CEA) comprising a        CDR-H1 comprising the amino acid sequence of SEQ ID NO:180, a        CDR-H2 comprising the amino acid sequence of SEQ ID NO:181, and        a CDR-H3 comprising the amino acid sequence of SEQ ID NO:182,        and a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:183, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:184, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:185; or    -   (iii) a heavy chain variable region (V_(H)CEA) comprising a        CDR-H1 comprising the amino acid sequence of SEQ ID NO:127, a        CDR-H2 comprising the amino acid sequence of SEQ ID NO:128, and        a CDR-H3 comprising the amino acid sequence of SEQ ID NO:129,        and a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:130, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:131, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:132, or    -   (iv) a heavy chain variable region (V_(H)CEA) comprising a        CDR-H1 comprising the amino acid sequence of SEQ ID NO:507, a        CDR-H2 comprising the amino acid sequence of SEQ ID NO:508, and        a CDR-H3 comprising the amino acid sequence of SEQ ID NO:509,        and a light chain variable region (V_(L)CEA) comprising a CDR-L1        comprising the amino acid sequence of SEQ ID NO:510, a CDR-L2        comprising the amino acid sequence of SEQ ID NO:511, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:512.

In one particular aspect, the antigen binding domain capable of specificbinding to CEA comprises a heavy chain variable region (V_(H)CEA)comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:188,a CDR-H2 comprising the amino acid sequence of SEQ ID NO:189, and aCDR-H3 comprising the amino acid sequence of SEQ ID NO:190, and a lightchain variable region (V_(L)CEA) comprising a CDR-L1 comprising theamino acid sequence of SEQ ID NO:191, a CDR-L2 comprising the amino acidsequence of SEQ ID NO:192, and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:193.

Particularly, the antigen binding domain capable of specific binding toCEA comprises a heavy chain variable region (V_(H)CEA) comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:133, and a lightchain variable region (V_(L)CEA) comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:134. In one aspect, the antigen bindingdomain capable of specific binding to CEA comprises a heavy chainvariable region (V_(H)CEA) comprising an amino acid sequence of SEQ IDNO:133, and a light chain variable region (V_(L)CEA) comprising theamino acid sequence of SEQ ID NO:134.

In another aspect, the antigen binding domain capable of specificbinding to CEA comprises a heavy chain variable region (V_(H)CEA)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:186,and a light chain variable region (V_(L)CEA) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:187. In one aspect,the antigen binding domain capable of specific binding to CEA comprisesa heavy chain variable region (V_(H)CEA) comprising an amino acidsequence of SEQ ID NO:186, and a light chain variable region (V_(L)CEA)comprising the amino acid sequence of SEQ ID NO:187.

In another aspect, the antigen binding domain capable of specificbinding to CEA comprises a heavy chain variable region (V_(H)CEA)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:513,and a light chain variable region (V_(L)CEA) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:514. In one aspect,the antigen binding domain capable of specific binding to CEA comprisesa heavy chain variable region (V_(H)CEA) comprising an amino acidsequence of SEQ ID NO:513, and a light chain variable region (V_(L)CEA)comprising the amino acid sequence of SEQ ID NO:514.

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule, wherein the antigen binding domain capable of specificbinding to CEA comprises

-   -   (a) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:194 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:195, or    -   (b) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:196 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:197, or    -   (c) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:198 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:199, or    -   (d) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:200 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:201, or    -   (e) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:202 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:203, or    -   (f) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:204 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:205, or    -   (g) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:206 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:207, or    -   (h) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:208 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:209, or    -   (i) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:210 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:211, or    -   (j) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:212 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:213.

Particularly, the antigen binding domain capable of specific binding toCEA comprises a heavy chain variable region (V_(H)CEA) comprising theamino acid sequence of SEQ ID NO:200 and a light chain variable region(V_(L)CEA) comprising the amino acid sequence of SEQ ID NO:201.

FAP-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to Fibroblast Activation Protein (FAP).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to FAP comprises

-   -   (a) a heavy chain variable region (V_(H)FAP) comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:13,        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:14, and a light chain variable region (V_(L)FAP)        comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ        ID NO:15, (v) CDR-L2 comprising the amino acid sequence of SEQ        ID NO:16, and (vi) CDR-L3 comprising the amino acid sequence of        SEQ ID NO:17, or    -   (b) a heavy chain variable region (V_(H)FAP) comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:6, and a light chain variable region (V_(L)FAP)        comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ        ID NO:7, (v) CDR-L2 comprising the amino acid sequence of SEQ ID        NO:8, and (vi) CDR-L3 comprising the amino acid sequence of SEQ        ID NO:9.

In particular, the antigen binding domain capable of specific binding toFAP comprises a heavy chain variable region (V_(H)FAP) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:13, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:14, and a light chainvariable region (V_(L)FAP) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:15, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:16, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:17. In one aspect, a bispecific agonistic CD28antigen binding molecule is provided, wherein the antigen binding domaincapable of specific binding to FAP comprises (a) a heavy chain variableregion (V_(H)FAP) comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:18, and a light chain variable region (V_(L)FAP)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:19,or (b) a heavy chain variable region (V_(H)FAP) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:10, and a light chainvariable region (V_(L)FAP) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:11. Particularly, the antigen binding domaincapable of specific binding to FAP comprises a heavy chain variableregion (V_(H)FAP) comprising the amino acid sequence of SEQ ID NO:18 anda light chain variable region (V_(L)FAP) comprising the amino acidsequence of SEQ ID NO:19.

EpCAM-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to epithelial cell adhesion molecule(EpCAM).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to EpCAM comprises a heavy chain variable region(V_(H)EpCAM) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:515, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:516, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:517, and a light chain variable region (V_(L)EpCAM) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:518, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:519, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:520. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to EpCAMcomprises a heavy chain variable region (V_(H)EpCAM) comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:521, and a light chainvariable region (V_(L)EpCAM) comprising an amino acid sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:522. Particularly, the antigen binding domaincapable of specific binding to EpCAM comprises a heavy chain variableregion (V_(H)EpCAM) comprising the amino acid sequence of SEQ ID NO:521and a light chain variable region (V_(L)EpCAM) comprising the amino acidsequence of SEQ ID NO:522.

HER3-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to HER3.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to HER3 comprises a heavy chain variable region(V_(H)HER3) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:523, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:524, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:525, and a light chain variable region (V_(L)HER3) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:526, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:527, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:528. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to HER3 comprisesa heavy chain variable region (V_(H)HER3) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:529, and a light chainvariable region (V_(L)HER3) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:530. Particularly, the antigen binding domaincapable of specific binding to HER3 comprises a heavy chain variableregion (V_(H)HER3) comprising the amino acid sequence of SEQ ID NO:529and a light chain variable region (V_(L)HER3) comprising the amino acidsequence of SEQ ID NO:530.

CD30-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to CD30.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to CD30 comprises a heavy chain variable region(V_(H)CD30) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:531, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:532, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:533, and a light chain variable region (V_(L)CD30) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:534, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:535, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:536. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to CD30 comprisesa heavy chain variable region (V_(H)CD30) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:537, and a light chainvariable region (V_(L)CD30) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:538. Particularly, the antigen binding domaincapable of specific binding to CD30 comprises a heavy chain variableregion (V_(H)CD30) comprising the amino acid sequence of SEQ ID NO:537and a light chain variable region (V_(L)CD30) comprising the amino acidsequence of SEQ ID NO:538.

TBPG (5T4)-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to TBPG (5T4).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to TBPG comprises a heavy chain variable region(V_(H)TBPG) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:539, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:540, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:541, and a light chain variable region (V_(L)TBPG) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:542, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:543, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:544. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to TBPG comprisesa heavy chain variable region (V_(H)TBPG) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:545, and a light chainvariable region (V L TBPG) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:546. Particularly, the antigen binding domaincapable of specific binding to TBPG comprises a heavy chain variableregion (V_(H)TBPG) comprising the amino acid sequence of SEQ ID NO:545and a light chain variable region (V_(L)TBPG) comprising the amino acidsequence of SEQ ID NO:546.

MM-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

The invention also provides novel bispecific agonistic CD28 antigenbinding molecules that are particularly useful in the treatment ofmultiple myeloma. The molecules comprise at least one antigen bindingdomain capable of specific binding to a Multiple Myeloma (MM) cellsurface antigen which causes cross-linking in the presence of MM cellsurface antigen-expressing cells and an Fc domain composed of a firstand a second subunit capable of stable association comprising one ormore amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or effector function (Fcsilent). Thus unspecific cross-linking via Fc receptors is avoided andspecific T cell activation in the presence of MM cell surfaceantigen-expressing cells is achieved.

Thus, herein provided is a bispecific agonistic CD28 antigen bindingmolecule comprising an antigen binding domain capable of specificbinding to CD28, an antigen binding domain capable of specific bindingto a Multiple Myeloma (MM) cell surface antigen, and a Fc domaincomposed of a first and a second subunit capable of stable associationcomprising one or more amino acid substitution that reduces the bindingaffinity of the antigen binding molecule to an Fc receptor and/oreffector function. In one aspect, the bispecific agonistic CD28 antigenbinding molecule as described herein is characterized by monovalentbinding to CD28. In a further aspect, the bispecific agonistic CD28antigen binding molecule as described herein is characterized bymonovalent binding to the Multiple Myeloma (MM) cell surface antigen.

In one aspect, a bispecific agonistic CD28 antigen binding molecule asdefined herein before is provided, wherein the Fc domain is an IgG,particularly an IgG1 Fc domain or an IgG4 Fc domain. In one particularaspect, the Fc domain composed of a first and a second subunit capableof stable association is an IgG1 Fc domain. The Fc domain comprises oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or reduces or abolisheseffector function. In one aspect, the Fc domain comprises the amino acidsubstitutions L234A and L235A (numbering according to Kabat EU index).In one aspect, the Fc domain is of human IgG1 subclass and comprises theamino acid mutations L234A, L235A and P329G (numbering according toKabat EU index).

In one aspect, a bispecific agonistic CD28 antigen binding molecule asdefined herein before is provided, wherein the MM cell surface antigenis selected from the group consisting of CD38, BCMA and GPRC5D.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to CD38.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to CD38 comprises a heavy chain variable region(V_(H)CD38) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:547, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:548, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:549, and a light chain variable region (V_(L)CD38) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:550, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:551, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:552. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to CD38 comprisesa heavy chain variable region (V_(H)CD38) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:553, and a light chainvariable region (V_(L)CD38) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:554. Particularly, the antigen binding domaincapable of specific binding to CD38 comprises a heavy chain variableregion (V_(H)CD38) comprising the amino acid sequence of SEQ ID NO:553and a light chain variable region (V_(L)CD38) comprising the amino acidsequence of SEQ ID NO:554.

In yet another aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to a tumor-associated antigen is an antigen bindingdomain capable of specific binding to BCMA.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to BCMA comprises a heavy chain variable region(V_(H)BCMA) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:555, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:556, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:557, and a light chain variable region (V_(L)BCMA) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:558, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:559, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:560. In one aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to BCMA comprisesa heavy chain variable region (V_(H)BCMA) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:561, and a light chainvariable region (V L BCMA) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:562. Particularly, the antigen binding domaincapable of specific binding to BCMA comprises a heavy chain variableregion (V_(H)BCMA) comprising the amino acid sequence of SEQ ID NO:561and a light chain variable region (V_(L)BCMA) comprising the amino acidsequence of SEQ ID NO:562.

GPRC5D-Targeting Bispecific Agonistic CD28 Antigen Binding Molecules

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a tumor-associated antigen is an antigen binding domaincapable of specific binding to GPRC5D.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to GPRC5D comprises a heavy chain variable region(V_(H)GPRC5D) comprising (i) CDR-H1 comprising the amino acid sequenceof SEQ ID NO:563, (ii) CDR-H2 comprising the amino acid sequence of SEQID NO:564, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:565, and a light chain variable region (V_(L)GPRC5D) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:566, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:567, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:568.

In one aspect, a bispecific agonistic CD28 antigen binding molecule isprovided, wherein the antigen binding domain capable of specific bindingto GPRC5D comprises a heavy chain variable region (V_(H)GPRC5D)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:569, SEQ ID NO:571, SEQ ID NO:572 and SEQ ID NO:573 and alight chain variable region (V L GPRC5D) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:570, SEQ IDNO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577 and SEQ ID NO:578.

In one aspect, a bispecific agonistic CD28 antigen binding molecule isprovided, wherein the antigen binding domain capable of specific bindingto GPRC5D comprises a heavy chain variable region (V_(H)GPRC5D)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:569,and a light chain variable region (V L GPRC5D) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:570. Particularly, theantigen binding domain capable of specific binding to GPRC5D comprises aheavy chain variable region (V_(H)GPRC5D) comprising the amino acidsequence of SEQ ID NO:569 and a light chain variable region (V L GPRC5D)comprising the amino acid sequence of SEQ ID NO:570.

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, wherein the antigen binding domaincapable of specific binding to GPRC5D comprises a heavy chain variableregion (V_(H)GPRC5D) comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:579, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:580, and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO:581, and a light chain variable region(V_(L)GPRC5D) comprising (iv) CDR-L1 comprising the amino acid sequenceof SEQ ID NO:582, (v) CDR-L2 comprising the amino acid sequence of SEQID NO:583, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:584.

In one aspect, a bispecific agonistic CD28 antigen binding molecule isprovided, wherein the antigen binding domain capable of specific bindingto GPRC5D comprises a heavy chain variable region (V_(H)GPRC5D)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 585, SEQ ID NO: 586, SEQ ID NO: 587, SEQ ID NO: 588, SEQ IDNO: 589 and SEQ ID NO:590 and a light chain variable region (V L GPRC5D)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 594 and SEQID NO: 595.

In another aspect, provided is bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto GPRC5D comprises

-   -   (a) a heavy chain variable region (V_(H)GPRC5D) comprising the        amino acid sequence of SEQ ID NO:569 and a light chain variable        region (V L GPRC5D) comprising the amino acid sequence of SEQ ID        NO:570, or    -   (b) a heavy chain variable region (V_(H)GPRC5D) comprising the        amino acid sequence of SEQ ID NO:573 and a light chain variable        region (V_(L)GPRC5D) comprising the amino acid sequence of SEQ        ID NO:576, or    -   (c) a heavy chain variable region (V_(H)GPRC5D) comprising the        amino acid sequence of SEQ ID NO:569 and a light chain variable        region (V L GPRC5D) comprising the amino acid sequence of SEQ ID        NO:572, or    -   (d) a heavy chain variable region (V_(H)GPRC5D) comprising the        amino acid sequence of SEQ ID NO:586 and a light chain variable        region (V L GPRC5D) comprising the amino acid sequence of SEQ ID        NO:593, or    -   (e) a heavy chain variable region (V_(H)GPRC5D) comprising the        amino acid sequence of SEQ ID NO:587 and a light chain variable        region (V L GPRC5D) comprising the amino acid sequence of SEQ ID        NO:592.

Bispecific Agonistic CD28 Antigen Binding Molecules Monovalent forBinding to CD28 and Monovalent for Binding to the Tumor-AssociatedAntigen (1+1 Format)

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to CEA,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a first light chain comprising theamino acid sequence of SEQ ID NO:65, a first heavy chain comprising theamino acid sequence of SEQ ID NO:66, a second heavy chain comprising theamino acid sequence of SEQ ID NO:87 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:88 (Molecule M).

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to FAP,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a first light chain comprising theamino acid sequence of SEQ ID NO:65, a first heavy chain comprising theamino acid sequence of SEQ ID NO:66, a second heavy chain comprising theamino acid sequence of SEQ ID NO:67 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:68 (Molecule C).

In a further aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second Fab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of one of        the Fc domain subunits.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a first light chain comprising theamino acid sequence of SEQ ID NO:77, a second light chain comprising theamino acid sequence of SEQ ID NO:78, a first heavy chain comprising theamino acid sequence of SEQ ID NO:75, and a second heavy chain comprisingthe amino acid sequence of SEQ ID NO:79 (Molecule H).

In a further aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) a Fab fragment capable of specific binding to CD28,    -   (b) a VH and VL domain capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,        wherein the Fab fragment capable of specific binding to CD28 is        fused at its C-terminus to the N-terminus of the first Fc domain        subunit, and wherein one of the VH and VL domain capable of        specific binding to a tumor-associated antigen is fused via a        peptide linker to the C-terminus of the first Fc domain subunit        and the other one of the VH and VL domain capable of specific        binding to a tumor-associated antigen is fused via a peptide        linker to the C-terminus of the second Fc domain subunit.

In one aspect, the peptide linker comprises an amino acid sequenceselected from SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:151 and SEQ IDNO:152. More particularly, the peptide linker comprises the SEQ IDNO:152.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a light chain comprising the aminoacid sequence of SEQ ID NO:62, a first heavy chain comprising the aminoacid sequence of SEQ ID NO:72, and a second heavy chain comprising theamino acid sequence of SEQ ID NO:80 (Molecule I).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54,        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to CEA        comprising        (i) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:186 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:187, or        (ii) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:200 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:201, or        (iii) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:513 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:514,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule comprising a first light chain comprising the amino acidsequence of SEQ ID NO:352, a first heavy chain comprising the amino acidsequence of SEQ ID NO:351, a second heavy chain comprising the aminoacid sequence of SEQ ID NO:353 and a second light chain comprising theamino acid sequence of SEQ ID NO:354 (Molecule 11A). In one aspect,provided is a bispecific agonistic CD28 antigen binding moleculecomprising a first light chain comprising the amino acid sequence of SEQID NO:352, a first heavy chain comprising the amino acid sequence of SEQID NO:351, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:355 and a second light chain comprising the amino acidsequence of SEQ ID NO:356 (Molecule 11B). In one aspect, provided is abispecific agonistic CD28 antigen binding molecule comprising a firstlight chain comprising the amino acid sequence of SEQ ID NO:352, a firstheavy chain comprising the amino acid sequence of SEQ ID NO:351, asecond heavy chain comprising the amino acid sequence of SEQ ID NO:357and a second light chain comprising the amino acid sequence of SEQ IDNO:358 (Molecule 11C). In one aspect, provided is a bispecific agonisticCD28 antigen binding molecule comprising a first light chain comprisingthe amino acid sequence of SEQ ID NO:352, a first heavy chain comprisingthe amino acid sequence of SEQ ID NO:351, a second heavy chaincomprising the amino acid sequence of SEQ ID NO:359 and a second lightchain comprising the amino acid sequence of SEQ ID NO:354 (Molecule11D). In one aspect, provided is a bispecific agonistic CD28 antigenbinding molecule comprising a first light chain comprising the aminoacid sequence of SEQ ID NO:370, a first heavy chain comprising the aminoacid sequence of SEQ ID NO:369, a second heavy chain comprising theamino acid sequence of SEQ ID NO:353 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:356 (Molecule 111). In one aspect,provided is a bispecific agonistic CD28 antigen binding moleculecomprising a first light chain comprising the amino acid sequence of SEQID NO:370, a first heavy chain comprising the amino acid sequence of SEQID NO:369, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:359 and a second light chain comprising the amino acidsequence of SEQ ID NO:354 (Molecule 11J). In one aspect, provided is abispecific agonistic CD28 antigen binding molecule comprising a firstlight chain comprising the amino acid sequence of SEQ ID NO:370, a firstheavy chain comprising the amino acid sequence of SEQ ID NO:369, asecond heavy chain comprising the amino acid sequence of SEQ ID NO:357and a second light chain comprising the amino acid sequence of SEQ IDNO:358 (Molecule 11K). In one aspect, provided is a bispecific agonisticCD28 antigen binding molecule comprising a first light chain comprisingthe amino acid sequence of SEQ ID NO:370, a first heavy chain comprisingthe amino acid sequence of SEQ ID NO:369, a second heavy chaincomprising the amino acid sequence of SEQ ID NO:359 and a second lightchain comprising the amino acid sequence of SEQ ID NO:356 (Molecule11L). In one aspect, the bispecific agonistic CD28 antigen bindingmolecule comprises a first light chain comprising the amino acidsequence of SEQ ID NO:376, a first heavy chain comprising the amino acidsequence of SEQ ID NO:375, a second heavy chain comprising the aminoacid sequence of SEQ ID NO:357 and a second light chain comprising theamino acid sequence of SEQ ID NO:358 (Molecule 11R). In one aspect, thebispecific agonistic CD28 antigen binding molecule comprises a firstlight chain comprising the amino acid sequence of SEQ ID NO:376, a firstheavy chain comprising the amino acid sequence of SEQ ID NO:375, asecond heavy chain comprising the amino acid sequence of SEQ ID NO:355and a second light chain comprising the amino acid sequence of SEQ IDNO:356 (Molecule 11S). In one aspect, the bispecific agonistic CD28antigen binding molecule comprises a first light chain comprising theamino acid sequence of SEQ ID NO:376, a first heavy chain comprising theamino acid sequence of SEQ ID NO:375, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO:355 and a second light chaincomprising the amino acid sequence of SEQ ID NO:354 (Molecule 11T).

In one particular aspect, the bispecific agonistic CD28 antigen bindingmolecule comprises a first light chain comprising the amino acidsequence of SEQ ID NO:376, a first heavy chain comprising the amino acidsequence of SEQ ID NO:375, a second heavy chain comprising the aminoacid sequence of SEQ ID NO:355 and a second light chain comprising theamino acid sequence of SEQ ID NO:356 (Molecule 11S). In anotherparticular aspect, provided is a bispecific agonistic CD28 antigenbinding molecule comprising a first light chain comprising the aminoacid sequence of SEQ ID NO:352, a first heavy chain comprising the aminoacid sequence of SEQ ID NO:351, a second heavy chain comprising theamino acid sequence of SEQ ID NO:355 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:356 (Molecule 11B).

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to CEA, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to CEA        comprising        (i) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:186 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:187, or        (ii) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:200 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:201, or        (iii) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:513 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:514,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:361, a first heavy chain comprising the amino acid sequence of SEQID NO:360, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:362 and a second light chain comprising the amino acidsequence of SEQ ID NO:363 (Molecule 11E). In one aspect, the bispecificagonistic CD28 antigen binding molecule comprises a first light chaincomprising the amino acid sequence of SEQ ID NO:361, a first heavy chaincomprising the amino acid sequence of SEQ ID NO:360, a second heavychain comprising the amino acid sequence of SEQ ID NO:364 and a secondlight chain comprising the amino acid sequence of SEQ ID NO:365(Molecule 11F). In one aspect, the bispecific agonistic CD28 antigenbinding molecule comprises a first light chain comprising the amino acidsequence of SEQ ID NO:361, a first heavy chain comprising the amino acidsequence of SEQ ID NO:360, a second heavy chain comprising the aminoacid sequence of SEQ ID NO:366 and a second light chain comprising theamino acid sequence of SEQ ID NO:367 (Molecule 11G). In one aspect, thebispecific agonistic CD28 antigen binding molecule comprises a firstlight chain comprising the amino acid sequence of SEQ ID NO:361, a firstheavy chain comprising the amino acid sequence of SEQ ID NO:360, asecond heavy chain comprising the amino acid sequence of SEQ ID NO:368and a second light chain comprising the amino acid sequence of SEQ IDNO:363 (Molecule 11H). In one aspect, the bispecific agonistic CD28antigen binding molecule comprises a first light chain comprising theamino acid sequence of SEQ ID NO:372, a first heavy chain comprising theamino acid sequence of SEQ ID NO:371, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO:368 and a second light chaincomprising the amino acid sequence of SEQ ID NO:363 (Molecule 11M). Inone aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:372, a first heavy chain comprising the amino acid sequence of SEQID NO:371, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:366 and a second light chain comprising the amino acidsequence of SEQ ID NO:367 (Molecule 11N). In one aspect, the bispecificagonistic CD28 antigen binding molecule comprises a first light chaincomprising the amino acid sequence of SEQ ID NO:372, a first heavy chaincomprising the amino acid sequence of SEQ ID NO:371, a second heavychain comprising the amino acid sequence of SEQ ID NO:364 and a secondlight chain comprising the amino acid sequence of SEQ ID NO:365(Molecule 110).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to EpCAM,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to EpCAM        comprising a heavy chain variable region (V_(H)EpCAM) comprising        the amino acid sequence of SEQ ID NO:521 and a light chain        variable region (V_(L)EpCAM) comprising the amino acid sequence        of SEQ ID NO:522,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to EpCAM, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to EpCAM        comprising a heavy chain variable region (V_(H)EpCAM) comprising        the amino acid sequence of SEQ ID NO:521 and a light chain        variable region (V_(L)EpCAM) comprising the amino acid sequence        of SEQ ID NO:522,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:367, a first heavy chain comprising the amino acid sequence of SEQID NO:366, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:390 and a second light chain comprising the amino acidsequence of SEQ ID NO:391 (Molecule 14A).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to HER3,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to HER3        comprising a heavy chain variable region (V_(H)HER3) comprising        the amino acid sequence of SEQ ID NO:529 and a light chain        variable region (V_(L)HER3) comprising the amino acid sequence        of SEQ ID NO:530,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:357, a first heavy chain comprising the amino acid sequence of SEQID NO:358, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:392 and a second light chain comprising the amino acidsequence of SEQ ID NO:393 (Molecule 14B).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to HER3, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to HER3        comprising a heavy chain variable region (V_(H)HER3) comprising        the amino acid sequence of SEQ ID NO:529 and a light chain        variable region (V_(L)HER3) comprising the amino acid sequence        of SEQ ID NO:530,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to CD30,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to CD30        comprising a heavy chain variable region (V_(H)CD30) comprising        the amino acid sequence of SEQ ID NO:537 and a light chain        variable region (V_(L)CD30) comprising the amino acid sequence        of SEQ ID NO:538,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:357, a first heavy chain comprising the amino acid sequence of SEQID NO:358, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:394 and a second light chain comprising the amino acidsequence of SEQ ID NO:395 (Molecule 14C).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to CD30, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to CD30        comprising a heavy chain variable region (V_(H)CD30) comprising        the amino acid sequence of SEQ ID NO:537 and a light chain        variable region (V_(L)CD30) comprising the amino acid sequence        of SEQ ID NO:538,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to TPBG,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to TPBG        comprising a heavy chain variable region (V_(H)TPBG) comprising        the amino acid sequence of SEQ ID NO:545 and a light chain        variable region (V_(L)TPBG) comprising the amino acid sequence        of SEQ ID NO:546,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:357, a first heavy chain comprising the amino acid sequence of SEQID NO:358, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:396 and a second light chain comprising the amino acidsequence of SEQ ID NO:397 (Molecule 14D).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to TPBG, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to TPBG        comprising a heavy chain variable region (V_(H)TPBG) comprising        the amino acid sequence of SEQ ID NO:545 and a light chain        variable region (V_(L)TPBG) comprising the amino acid sequence        of SEQ ID NO:546,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to CD38,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to CD38        comprising a heavy chain variable region (V_(H)CD38) comprising        the amino acid sequence of SEQ ID NO:553 and a light chain        variable region (V_(L)CD38) comprising the amino acid sequence        of SEQ ID NO:554,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:357, a first heavy chain comprising the amino acid sequence of SEQID NO:358, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:400 and a second light chain comprising the amino acidsequence of SEQ ID NO:401 (Molecule 16C).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to CD38, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to CD38        comprising a heavy chain variable region (V_(H)CD38) comprising        the amino acid sequence of SEQ ID NO:553 and a light chain        variable region (V_(L)CD38) comprising the amino acid sequence        of SEQ ID NO:554,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to BCMA,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to BCMA        comprising a heavy chain variable region (V_(H)BCMA) comprising        the amino acid sequence of SEQ ID NO:561 and a light chain        variable region (V_(L)BCMA) comprising the amino acid sequence        of SEQ ID NO:562,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to BCMA, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to BCMA        comprising a heavy chain variable region (V_(H)BCMA) comprising        the amino acid sequence of SEQ ID NO:561 and a light chain        variable region (V_(L)BCMA) comprising the amino acid sequence        of SEQ ID NO:562,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:367, a first heavy chain comprising the amino acid sequence of SEQID NO:366, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:402 and a second light chain comprising the amino acidsequence of SEQ ID NO:403 (Molecule 16D).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to GPRC5D,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to GPRC5D        comprising a heavy chain variable region (VHGPRC5D) comprising        the amino acid sequence of SEQ ID NO:569 and a light chain        variable region (VLGPRC5D) comprising the amino acid sequence of        SEQ ID NO:570,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:365, a first heavy chain comprising the amino acid sequence of SEQID NO:364, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:398 and a second light chain comprising the amino acidsequence of SEQ ID NO:399 (Molecule 16B).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to GPRC5D, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to GPRC5D        comprising a heavy chain variable region (V_(H)GPRC5D)        comprising the amino acid sequence of SEQ ID NO:569 and a light        chain variable region (V_(L)GPRC5D) comprising the amino acid        sequence of SEQ ID NO:570,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises a first light chain comprising the amino acid sequence of SEQID NO:367, a first heavy chain comprising the amino acid sequence of SEQID NO:366, a second heavy chain comprising the amino acid sequence ofSEQ ID NO:398 and a second light chain comprising the amino acidsequence of SEQ ID NO:399 (Molecule 16A).

Bispecific Agonistic CD28 Antigen Binding Molecules Monovalent forBinding to CD28 and Bivalent for Binding to the Tumor-Associated Antigen(1+2 Format)

In another aspect, a bispecific agonistic CD28 antigen binding moleculeas disclosed herein is provided, comprising

-   -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second and a third Fab fragment capable of specific        binding to a tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of the        first Fc domain subunit, and the third Fab fragment capable of        specific binding to a tumor-associated antigen is fused at the        C-terminus of the Fab heavy chain to the N-terminus of the        second Fc domain subunit.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising two light chains, each comprisingthe amino acid sequence of SEQ ID NO:78, one light chain comprising theamino acid sequence of SEQ ID NO:77, a first heavy chain comprising theamino acid sequence of SEQ ID NO:75, and a second heavy chain comprisingthe amino acid sequence of SEQ ID NO:76 (Molecule G).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) a first crossFab fragment capable of specific binding to        CD28 comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) a second and a third Fab fragment capable of specific        binding to fragment capable of specific binding to CEA        comprising        (i) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:186 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:187, or        (ii) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:200 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:201, or        (iii) a heavy chain variable region (V_(H)CEA) comprising the        amino acid sequence of SEQ ID NO:513 and a light chain variable        region (V_(L)CEA) comprising the amino acid sequence of SEQ ID        NO:514,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising two light chains, each comprisingthe amino acid sequence of SEQ ID NO:361, one light chain comprising theamino acid sequence of SEQ ID NO:368, a first heavy chain comprising theamino acid sequence of SEQ ID NO:362, and a second heavy chaincomprising the amino acid sequence of SEQ ID NO:373 (Molecule 11P).

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising two light chains, each comprisingthe amino acid sequence of SEQ ID NO:361, one light chain comprising theamino acid sequence of SEQ ID NO:368, a first heavy chain comprising theamino acid sequence of SEQ ID NO:360, and a second heavy chaincomprising the amino acid sequence of SEQ ID NO:374 (Molecule 11Q).

FAP- and CEA-Targeting Agonistic CD28 Antigen Binding Molecules

Herein provided is also a bispecific agonistic CD28 antigen bindingmolecule with monovalent binding to CD28, comprising

-   -   (a) one antigen binding domains capable of specific binding to        CD28,    -   (b) one antigen binding domain capable of specific binding to a        first tumor-associated antigen, and    -   (c) an Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,        characterized in that it additionally comprises one antigen        binding domain capable of specific binding to a second        tumor-associated antigen.

In one particular aspect, provided is a trispecific agonistic CD28antigen binding molecule with monovalent binding to CD28, comprising

-   -   (a) one antigen binding domains capable of specific binding to        CD28,    -   (b) one antigen binding domain capable of specific binding to        CEA and one antigen binding domain capable of specific binding        to FAP,    -   (c) an Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a first light chain comprising theamino acid sequence of SEQ ID NO:88, a first heavy chain comprising theamino acid sequence of SEQ ID NO:87, a second heavy chain comprising theamino acid sequence of SEQ ID NO:388 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:389 (Molecule Y).

B Cell Surface Antigen-Targeting Bispecific Agonistic CD28 AntigenBinding Molecules

The invention provides novel bispecific agonistic CD28 antigen bindingmolecules with particularly advantageous properties such asproducibility, stability, binding affinity, biological activity,targeting efficiency, reduced toxicity, an extended dosage range thatcan be given to a patient and thereby a possibly enhanced efficacy. Thenovel bispecific agonistic CD28 antigen binding molecules comprise an Fcdomain composed of a first and a second subunit capable of stableassociation comprising one or more amino acid substitution that reducesthe binding affinity of the antigen binding molecule to an Fc receptorand/or effector function (Fc silent) and thus unspecific cross-linkingvia Fc receptors is avoided. Instead, they comprise at least one antigenbinding domain capable of specific binding to a B cell surface antigensuch as CD19 or CD79b which causes cross-linking in the presence ofCD19- or CD79b-expressing B cells. Thus, specific T cell activation inthe presence of CD19- or CD79b-expressing B cells is achieved.

Herein provided is a bispecific agonistic CD28 antigen binding moleculecomprising an antigen binding domain capable of specific binding toCD28, an antigen binding domain capable of specific binding to a B cellsurface antigen, and a Fc domain composed of a first and a secondsubunit capable of stable association comprising one or more amino acidsubstitution that reduces the binding affinity of the antigen bindingmolecule to an Fc receptor and/or effector function. In one aspect, thebispecific agonistic CD28 antigen binding molecule as described hereinis characterized by monovalent binding to CD28. In a further aspect, thebispecific agonistic CD28 antigen binding molecule as described hereinis characterized by monovalent binding to the B cell surface antigen.

In one aspect, a bispecific agonistic CD28 antigen binding molecule asdefined herein before is provided, wherein the Fc domain is an IgG,particularly an IgG1 Fc domain or an IgG4 Fc domain. In one particularaspect, the Fc domain composed of a first and a second subunit capableof stable association is an IgG1 Fc domain. The Fc domain comprises oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or reduces or abolisheseffector function. In one aspect, the Fc domain comprises the amino acidsubstitutions L234A and L235A (numbering according to Kabat EU index).In one aspect, the Fc domain is of human IgG1 subclass and comprises theamino acid mutations L234A, L235A and P329G (numbering according toKabat EU index).

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as defined herein before, wherein the antigen binding domaincapable of specific binding to CD28 comprises

-   -   (i) a heavy chain variable region (V_(H)CD28) comprising a heavy        chain complementary determining region CDR-H1 of SEQ ID NO: 20,        a CDR-H2 of SEQ ID NO: 21, and a CDR-H3 of SEQ ID NO: 22, and a        light chain variable region (V_(L)CD28) comprising a light chain        complementary determining region CDR-L1 of SEQ ID NO: 23, a        CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQ ID NO: 25; or    -   (ii) a heavy chain variable region (V_(H)CD28) comprising a        CDR-H1 of SEQ ID NO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3        of SEQ ID NO: 38, and a light chain variable region (V_(L)CD28)        comprising a CDR-L1 of SEQ ID NO: 39, a CDR-L2 of SEQ ID NO: 40        and a CDR-L3 of SEQ ID NO: 41.

In one aspect, the antigen binding domain capable of specific binding toCD28 of the bispecific agonistic CD28 antigen binding molecule comprisesa heavy chain variable region (V_(H)CD28) comprising a CDR-H1 of SEQ IDNO:20, a CDR-H2 of SEQ ID NO:21, and a CDR-H3 of SEQ ID NO:22, and alight chain variable region (V_(L)CD28) comprising a CDR-L1 of SEQ IDNO:23, a CDR-L2 of SEQ ID NO:24 and a CDR-L3 of SEQ ID NO:25. In oneaspect, provided is a bispecific agonistic CD28 antigen binding moleculeas defined herein before, wherein the antigen binding domain capable ofspecific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:26, and a light chain variable region (V_(L)CD28) comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO:27. In oneaspect, the antigen binding domain capable of specific binding to CD28comprises a heavy chain variable region (V_(H)CD28) comprising the aminoacid sequence of SEQ ID NO:26 and a light chain variable region(V_(L)CD28) comprising the amino acid sequence of SEQ ID NO:27.

In another aspect, the antigen binding domain capable of specificbinding to CD28 of the bispecific agonistic CD28 antigen bindingmolecule comprises a heavy chain variable region (V_(H)CD28) comprisinga CDR-H1 of SEQ ID NO: 28, a CDR-H2 of SEQ ID NO: 29, and a CDR-H3 ofSEQ ID NO: 30, and a light chain variable region (V_(L)CD28) comprisinga CDR-L1 of SEQ ID NO: 31, a CDR-L2 of SEQ ID NO:32 and a CDR-L3 of SEQID NO:33. In one aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as defined herein before, wherein the antigen bindingdomain capable of specific binding to CD28 comprises a heavy chainvariable region (V_(H)CD28) comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:34, and a light chain variable region (V_(L)CD28)comprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:35.In one aspect, the antigen binding domain capable of specific binding toCD28 comprises a heavy chain variable region (V_(H)CD28) comprising theamino acid sequence of SEQ ID NO:34 and a light chain variable region(V_(L)CD28) comprising the amino acid sequence of SEQ ID NO:

In another aspect, the antigen binding domain capable of specificbinding to CD28 of the bispecific agonistic CD28 antigen bindingmolecule comprises a heavy chain variable region (V_(H)CD28) comprisinga CDR-H1 of SEQ ID NO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3 ofSEQ ID NO: 38, and a light chain variable region (V_(L)CD28) comprisinga CDR-L1 of SEQ ID NO: 39, a CDR-L2 of SEQ ID NO: 40 and a CDR-L3 of SEQID NO: 41.

In a further aspect, a bispecific agonistic CD28 antigen bindingmolecule is provided, wherein the antigen binding domain capable ofspecific binding to CD28 comprises a heavy chain variable region(V_(H)CD28) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 andSEQ ID NO:51, and a light chain variable region (V_(L)CD28) comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:27, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ IDNO:61.

In another aspect, provided is bispecific agonistic CD28 antigen bindingmolecule, wherein the antigen binding domain capable of specific bindingto CD28 comprises

-   -   (a) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (b) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (c) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:51 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:61, or    -   (d) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (e) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of

SEQ ID NO:54, or

-   -   (f) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (g) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (h) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:43 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (j) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (k) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27.

In one aspect, the bispecific agonistic CD28 antigen binding moleculecomprises an antigen binding domain capable of specific binding to CD28comprising a heavy chain variable region (V_(H)CD28) comprising theamino acid sequence of SEQ ID NO:46 and a light chain variable region(V_(L)CD28) comprising the amino acid sequence of SEQ ID NO:53, or aheavy chain variable region (V_(H)CD28) comprising the amino acidsequence of SEQ ID NO:47 and a light chain variable region (V_(L)CD28)comprising the amino acid sequence of SEQ ID NO:54, or a heavy chainvariable region (V_(H)CD28) comprising the amino acid sequence of SEQ IDNO:47 and a light chain variable region (V_(L)CD28) comprising the aminoacid sequence of SEQ ID NO:27, or a heavy chain variable region(V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:42 and alight chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:27. In one particular aspect, a bispecificagonistic CD28 antigen binding molecule is provided, wherein the antigenbinding domain capable of specific binding to CD28 comprises a heavychain variable region (V_(H)CD28) comprising the amino acid sequence ofSEQ ID NO:46 and a light chain variable region (V_(L)CD28) comprisingthe amino acid sequence of SEQ ID NO:53. In another particular aspect, abispecific agonistic CD28 antigen binding molecule is provided, whereinthe antigen binding domain capable of specific binding to CD28 comprisesa heavy chain variable region (V_(H)CD28) comprising the amino acidsequence of SEQ ID NO:47 and a light chain variable region (V_(L)CD28)comprising the amino acid sequence of SEQ ID NO:54. In furtherparticular aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to CD28 comprises a heavy chain variable region (V_(H)CD28)comprising the amino acid sequence of SEQ ID NO:47 and a light chainvariable region (V_(L)CD28) comprising the amino acid sequence of SEQ IDNO:27. In yet another particular aspect, a bispecific agonistic CD28antigen binding molecule is provided, wherein the antigen binding domaincapable of specific binding to CD28 comprises a heavy chain variableregion (V_(H)CD28) comprising the amino acid sequence of SEQ ID NO:42and a light chain variable region (V_(L)CD28) comprising the amino acidsequence of SEQ ID NO:27.

In one aspect, a bispecific agonistic CD28 antigen binding molecule isprovided, wherein the antigen binding domain capable of specific bindingto B cell surface antigen is an antigen binding domain capable ofspecific binding to CD19.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to CD19 comprises (a) a heavy chain variable region(V_(H)CD19) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:406, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:407, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:408, and a light chain variable region (V_(L)CD19) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:409, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:410, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:411, or (b) a heavychain variable region (V_(H)CD19) comprising (i) CDR-H1 comprising theamino acid sequence of SEQ ID NO:414, (ii) CDR-H2 comprising the aminoacid sequence of SEQ ID NO:415, and (iii) CDR-H3 comprising the aminoacid sequence of SEQ ID NO:416, and a light chain variable region(V_(L)CD19) comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:417, (v) CDR-L2 comprising the amino acid sequence of SEQ IDNO:418, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:419. Particularly, the antigen binding domain capable of specificbinding to CD19 comprises (a) a heavy chain variable region (V_(H)CD19)comprising an amino acid sequence that is at least about 95%, 98% or100% identical to the amino acid sequence of SEQ ID NO:412, and a lightchain variable region (V_(L)CD19) comprising an amino acid sequence thatis at least about 95%, 98% or 100% identical to the amino acid sequenceof SEQ ID NO:413, or (b) a heavy chain variable region (V_(H)CD19)comprising an amino acid sequence that is at least about 95%, 98% or100% identical to the amino acid sequence of SEQ ID NO:420, and a lightchain variable region (V_(L)CD19) comprising an amino acid sequence thatis at least about 95%, 98% or 100% identical to the amino acid sequenceof SEQ ID NO:421. In one particular aspect, the antigen binding domaincapable of specific binding to CD19 comprises a heavy chain variableregion (V_(H)CD19) comprising an amino acid sequence of SEQ ID NO:412and a light chain variable region (V_(L)CD19) comprising an amino acidsequence of SEQ ID NO:413.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeis provided, wherein the antigen binding domain capable of specificbinding to a B cell surface antigen is an antigen binding domain capableof specific binding to CD79b.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, wherein the antigen binding domain capableof specific binding to CD79b comprises a heavy chain variable region(V_(H)CD79b) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:422, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:423, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:424, and a light chain variable region (V_(L)CD79b) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:425, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:426, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:427. In particular, theantigen binding domain capable of specific binding to CD79b comprises aheavy chain variable region (V_(H)CD79b) comprising an amino acidsequence that is at least about 95%, 98%, or 100% identical to the aminoacid sequence of SEQ ID NO:428, and a light chain variable region(V_(L)CD79b) comprising an amino acid sequence that is at least about95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO:429.In one aspect, the antigen binding domain capable of specific binding toCD79b comprises a heavy chain variable region (V_(H)CD79b) comprisingthe amino acid sequence of SEQ ID NO:428, and a light chain variableregion (V_(L)CD79b) comprising the amino acid sequence of SEQ ID NO:429.

In a further aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as defined herein before, wherein the antigen bindingdomain capable of specific binding to CD28 is a Fab fragment or acrossFab fragment. In one particular aspect, the antigen binding domaincapable of specific binding to CD28 is a Fab fragment and the antigenbinding domain capable of specific binding to a B cell surface antigenis a crossFab fragment.

Bispecific Agonistic CD28 Antigen Binding Molecules Monovalent forBinding to CD28 and Monovalent for Binding to a B Cell Surface Antigen(1+1 Format)

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to a B        cell surface antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to CD19,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to CD19        comprising        (i) a heavy chain variable region (V_(H)CD19) comprising the        amino acid sequence of SEQ ID NO:412 and a light chain variable        region (V_(L)CD19) comprising the amino acid sequence of SEQ ID        NO:413, or        (ii) a heavy chain variable region (V_(H)CD19) comprising the        amino acid sequence of SEQ ID NO:420 and a light chain variable        region (V_(L)CD19) comprising the amino acid sequence of SEQ ID        NO:421,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule comprising a first light chain comprising the amino acidsequence of SEQ ID NO:65, a first heavy chain comprising the amino acidsequence of SEQ ID NO:118, a second heavy chain comprising the aminoacid sequence of SEQ ID NO:430 and a second light chain comprising theamino acid sequence of SEQ ID NO:431 (Molecule 18A).

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a first light chain comprising theamino acid sequence of SEQ ID NO:121, a first heavy chain comprising theamino acid sequence of SEQ ID NO:116, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO:430 and a second light chaincomprising the amino acid sequence of SEQ ID NO:431 (Molecule 18B).

In another particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a first light chain comprising theamino acid sequence of SEQ ID NO:122, a first heavy chain comprising theamino acid sequence of SEQ ID NO:114, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO:430 and a second light chaincomprising the amino acid sequence of SEQ ID NO:431 (Molecule 18C).

In one further aspect, provided is a bispecific agonistic CD28 antigenbinding molecule comprising a first light chain comprising the aminoacid sequence of SEQ ID NO:65, a first heavy chain comprising the aminoacid sequence of SEQ ID NO:114, a second heavy chain comprising theamino acid sequence of SEQ ID NO:430 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:431 (Molecule 18D).

In yet another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule comprising a first light chain comprising the aminoacid sequence of SEQ ID NO:123, a first heavy chain comprising the aminoacid sequence of SEQ ID NO:118, a second heavy chain comprising theamino acid sequence of SEQ ID NO:430 and a second light chain comprisingthe amino acid sequence of SEQ ID NO:431 (Molecule 18E).

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to CD19, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of specific binding to CD19        comprising        (i) a heavy chain variable region (V_(H)CD19) comprising the        amino acid sequence of SEQ ID NO:412 and a light chain variable        region (V_(L)CD19) comprising the amino acid sequence of SEQ ID        NO:413, or        (ii) a heavy chain variable region (V_(H)CD19) comprising the        amino acid sequence of SEQ ID NO:420 and a light chain variable        region (V_(L)CD19) comprising the amino acid sequence of SEQ ID        NO:421,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to CD79b,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one Fab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one crossFab fragment capable of specific binding to CD79b        comprising a heavy chain variable region (V_(H)CD79b) comprising        the amino acid sequence of SEQ ID NO:428 and a light chain        variable region (V_(L)CD79b) comprising the amino acid sequence        of SEQ ID NO:429,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one particular aspect, provided is a bispecific agonistic CD28antigen binding molecule comprising a first light chain comprising theamino acid sequence of SEQ ID NO:121, a first heavy chain comprising theamino acid sequence of SEQ ID NO:116, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO:432 and a second light chaincomprising the amino acid sequence of SEQ ID NO:433 (Molecule 18F).

In another aspect, provided is a bispecific agonistic CD28 antigenbinding molecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28,    -   (b) one Fab fragment capable of specific binding to CD19, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

In one aspect, provided is a bispecific agonistic CD28 antigen bindingmolecule as described herein, comprising

-   -   (a) one crossFab fragment capable of specific binding to CD28        comprising        (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or        (ii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or        (iii) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27,    -   (b) one Fab fragment capable of of specific binding to CD79b        comprising a heavy chain variable region (V_(H)CD79b) comprising        the amino acid sequence of SEQ ID NO:428 and a light chain        variable region (V_(L)CD79b) comprising the amino acid sequence        of SEQ ID NO:429,    -   and (c) a Fc domain composed of a first and a second subunit        capable of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.

Bispecific Agonistic CD28 Antigen Binding Molecules Monovalent forBinding to CD28 and Bivalent for Binding to a B Cell Surface Antigen(1+2 Format)

In one aspect, the bispecific agonistic CD28 antigen binding molecule ischaracterized by bivalent binding to the B cell surface antigen.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeas disclosed herein is provided, comprising

-   -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second and a third Fab fragment capable of specific        binding to a B cell surface antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of the        first Fc domain subunit, and the third Fab fragment capable of        specific binding to a tumor-associated antigen is fused at the        C-terminus of the Fab heavy chain to the N-terminus of the        second Fc domain subunit.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain of the bispecific agonistic CD28 antigen binding moleculeof the invention consists of a pair of polypeptide chains comprisingheavy chain domains of an immunoglobulin molecule. For example, the Fcdomain of an immunoglobulin G (IgG) molecule is a dimer, each subunit ofwhich comprises the CH2 and CH3 IgG heavy chain constant domains. Thetwo subunits of the Fc domain are capable of stable association witheach other. The Fc domain confers favorable pharmacokinetic propertiesto the antigen binding molecules of the invention, including a longserum half-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. On the otherside, it may, however, lead to undesirable targeting of the bispecificantibodies of the invention to cells expressing Fc receptors rather thanto the preferred antigen-bearing cells.

Accordingly, the Fc domain of the bispecific agonistic CD28 antigenbinding molecule of the invention exhibits reduced binding affinity toan Fc receptor and/or reduced effector function, as compared to a nativeIgG1 Fc domain. In one aspect, the Fc does not substantially bind to anFc receptor and/or does not induce effector function. In a particularaspect the Fc receptor is an Fcγ receptor. In one aspect, the Fcreceptor is a human Fc receptor. In a specific aspect, the Fc receptoris an activating human Fcγ receptor, more specifically human FcγRIIIa,FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, theFc domain does not induce effector function. The reduced effectorfunction can include, but is not limited to, one or more of thefollowing: reduced complement dependent cytotoxicity (CDC), reducedantibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced dendritic cell maturation, or reduced T cell priming.

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In one particular aspect, the invention provides an antigen bindingmolecule, wherein the Fc region comprises one or more amino acidsubstitution that reduces binding to an Fc receptor, in particulartowards Fcγ receptor. In one aspect, the invention provides an antibody,wherein the Fc region comprises one or more amino acid substitution andwherein the ADCC induced by the antibody is reduced to 0-20% of the ADCCinduced by an antibody comprising the wild-type human IgG1 Fc region.

In one aspect, the Fc domain of the antigen binding molecule of theinvention comprises one or more amino acid mutation that reduces thebinding affinity of the Fc domain to an Fc receptor and/or effectorfunction. Typically, the same one or more amino acid mutation is presentin each of the two subunits of the Fc domain. In particular, the Fcdomain comprises an amino acid substitution at a position of E233, L234,L235, N297, P331 and P329 (EU numbering). In particular, the Fc domaincomprises amino acid substitutions at positions 234 and 235 (EUnumbering) and/or 329 (EU numbering) of the IgG heavy chains. Moreparticularly, provided is an antigen binding molecule according to theinvention which comprises an Fc domain with the amino acid substitutionsL234A, L235A and P329G (“P329G LALA”, EU numbering) in the IgG heavychains. The amino acid substitutions L234A and L235A refer to theso-called LALA mutation. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcγ receptor binding of ahuman IgG1 Fc domain and is described in International Patent Appl.Publ. No. WO 2012/130831 A1 which also describes methods of preparingsuch mutant Fc domains and methods for determining its properties suchas Fc receptor binding or effector functions.

Fc domains with reduced Fc receptor binding and/or effector functionalso include those with substitution of one or more of Fc domainresidues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).Such Fc mutants include Fc mutants with substitutions at two or more ofamino acid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

In another aspect, the Fc domain is an IgG4 Fc domain. IgG4 antibodiesexhibit reduced binding affinity to Fc receptors and reduced effectorfunctions as compared to IgG1 antibodies. In a more specific aspect, theFc domain is an IgG4 Fc domain comprising an amino acid substitution atposition 5228 (Kabat numbering), particularly the amino acidsubstitution S228P. In a more specific aspect, the Fc domain is an IgG4Fc domain comprising amino acid substitutions L235E and S228P and P329G(EU numbering). Such IgG4 Fc domain mutants and their Fcγ receptorbinding properties are also described in WO 2012/130831.

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. Alternatively, binding affinity ofFc domains or cell activating antibodies comprising an Fc domain for Fcreceptors may be evaluated using cell lines known to express particularFc receptors, such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or antigen binding molecules of theinvention comprising an Fc domain, can be measured by methods known inthe art. A suitable assay for measuring ADCC is described herein. Otherexamples of in vitro assays to assess ADCC activity of a molecule ofinterest are described in U.S. Pat. No. 5,500,362; Hellstrom et al. ProcNatl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc NatlAcad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemannet al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactiveassays methods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega,Madison, WI)). Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in an animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 652-656 (1998).

In some aspects, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some aspects whereinthe Fc domain is engineered to have reduced effector function, saidreduced effector function includes reduced CDC. C1q binding assays maybe carried out to determine whether the bispecific antibodies of theinvention are able to bind C1q and hence has CDC activity. See e.g., C1qand C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al.,Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743(2004)).

In one particular aspect, the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG1 Fc domain, is a human IgG1 Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG4 Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G (numberings according to Kabat EU index). Moreparticularly, it is a human IgG1 Fc domain comprising the amino acidsubstitutions L234A, L235A and P329G (numbering according to Kabat EUindex).

Fc Domain Modifications Promoting Heterodimerization

The bispecific agonistic CD28 antigen binding molecules of the inventioncomprise different antigen-binding sites, fused to one or the other ofthe two subunits of the Fc domain, thus the two subunits of the Fcdomain may be comprised in two non-identical polypeptide chains.Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of the bispecific antigenbinding molecules of the invention in recombinant production, it willthus be advantageous to introduce in the Fc domain of the bispecificantigen binding molecules of the invention a modification promoting theassociation of the desired polypeptides.

Accordingly, in particular aspects the invention relates to thebispecific agonistic CD28 antigen binding molecule with monovalentbinding to CD28 comprising (a) one antigen binding domain capable ofspecific binding to CD28, (b) at least one antigen binding domaincapable of specific binding to a tumor-associated antigen, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation comprising one or more amino acid substitution that reducesthe binding affinity of the antigen binding molecule to an Fc receptorand/or effector function, wherein the Fc domain comprises a modificationpromoting the association of the first and second subunit of the Fcdomain. The site of most extensive protein-protein interaction betweenthe two subunits of a human IgG Fc domain is in the CH3 domain of the Fcdomain. Thus, in one aspect said modification is in the CH3 domain ofthe Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole”modification, comprising a “knob” modification in one of the twosubunits of the Fc domain and a “hole” modification in the other one ofthe two subunits of the Fc domain. Thus, the invention relates to thebispecific agonistic CD28 antigen binding molecule with monovalentbinding to CD28 comprising (a) one antigen binding domain capable ofspecific binding to CD28, (b) at least one antigen binding domaincapable of specific binding to a tumor-associated antigen, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation comprising one or more amino acid substitution that reducesthe binding affinity of the antigen binding molecule to an Fc receptorand/or effector function, wherein the first subunit of the Fc domaincomprises knobs and the second subunit of the Fc domain comprises holesaccording to the knobs into holes method. In a particular aspect, thefirst subunit of the Fc domain comprises the amino acid substitutionsS354C and T366W (EU numbering) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in one aspect, in the CH3 domain of the first subunit ofthe Fc domain of the bispecific antigen binding molecules of theinvention an amino acid residue is replaced with an amino acid residuehaving a larger side chain volume, thereby generating a protuberancewithin the CH3 domain of the first subunit which is positionable in acavity within the CH3 domain of the second subunit, and in the CH3domain of the second subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity within the CH3 domain of the second subunitwithin which the protuberance within the CH3 domain of the first subunitis positionable. The protuberance and cavity can be made by altering thenucleic acid encoding the polypeptides, e.g. by site-specificmutagenesis, or by peptide synthesis. In a specific aspect, in the CH3domain of the first subunit of the Fc domain the threonine residue atposition 366 is replaced with a tryptophan residue (T366W), and in theCH3 domain of the second subunit of the Fc domain the tyrosine residueat position 407 is replaced with a valine residue (Y407V). In oneaspect, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A).

In yet a further aspect, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter (2001), J ImmunolMethods 248, 7-15). In a particular aspect, the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (EUnumbering) and the second subunit of the Fc domain comprises the aminoacid substitutions Y349C, T366S and Y407V (numbering according to KabatEU index).

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

The C-terminus of the heavy chain of the bispecific agonistic CD28antigen binding molecule as reported herein can be a complete C-terminusending with the amino acid residues PGK. The C-terminus of the heavychain can be a shortened C-terminus in which one or two of the Cterminal amino acid residues have been removed. In one preferred aspect,the C-terminus of the heavy chain is a shortened C-terminus ending P. Inone preferred aspect, the C-terminus of the heavy chain is a shortenedC-terminus ending PG. In one aspect of all aspects as reported herein, aCD28 antigen binding molecule comprising a heavy chain including aC-terminal CH3 domain as specified herein, comprises the C-terminalglycine-lysine dipeptide (G₄₄₆ and K447, numbering according to Kabat EUindex). In one aspect of all aspects as reported herein, a CD28 antigenbinding molecule comprising a heavy chain including a C-terminal CH3domain, as specified herein, comprises a C-terminal glycine residue(G₄₄₆, numbering according to Kabat EU index).

Modifications in the Fab Domains

In one aspect, the invention relates to a bispecific agonistic CD28antigen binding molecule characterized by monovalent binding to CD28comprising (a) one antigen binding domain capable of specific binding toCD28, (b) at least one antigen binding domain capable of specificbinding to a tumor-associated antigen, and (c) a Fc domain composed of afirst and a second subunit capable of stable association comprising oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or effector function,wherein the at least one antigen binding domain capable of specificbinding to a tumor-associated antigen is a Fab fragment and in the Fabfragment either the variable domains VH and VL or the constant domainsCH1 and CL are exchanged according to the Crossmab technology.

Multispecific antibodies with a domain replacement/exchange in onebinding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail inWO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. Theyclearly reduce the byproducts caused by the mismatch of a light chainagainst a first antigen with the wrong heavy chain against the secondantigen (compared to approaches without such domain exchange).

In one aspect, the invention relates to a bispecific agonistic CD28antigen binding molecule characterized by monovalent binding to CD28comprising (a) one antigen binding domain capable of specific binding toCD28, (b) at least one antigen binding domain capable of specificbinding to a tumor-associated antigen, and (c) a Fc domain composed of afirst and a second subunit capable of stable association comprising oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or effector function,wherein in the Fab fragments capable of specific binding to atumor-associated antigen the constant domains CL and CH1 are replaced byeach other so that the CH1 domain is part of the light chain and the CLdomain is part of the heavy chain.

In another aspect, and to further improve correct pairing, thebispecific agonistic CD28 antigen binding molecule characterized bymonovalent binding to CD28 comprising (a) one antigen binding domaincapable of specific binding to CD28, (b) at least one antigen bindingdomains capable of specific binding to a tumor-associated antigen, and(c) a Fc domain composed of a first and a second subunit capable ofstable association comprising one or more amino acid substitution thatreduces the binding affinity of the antigen binding molecule to an Fcreceptor and/or effector function, can contain different charged aminoacid substitutions (so-called “charged residues”). These modificationsare introduced in the crossed or non-crossed CH1 and CL domains. In aparticular aspect, the invention relates to a bispecific agonistic CD28antigen binding molecule, wherein in one of CL domains the amino acid atposition 123 (EU numbering) has been replaced by arginine (R) and theamino acid at position 124 (EU numbering) has been substituted by lysine(K) and wherein in one of the CH1 domains the amino acids at position147 (EU numbering) and at position 213 (EU numbering) have beensubstituted by glutamic acid (E). In one particular aspect, in the CLdomain of the Fab fragment capable of specific binding to CD28 the aminoacid at position 123 (EU numbering) has been replaced by arginine (R)and the amino acid at position 124 (EU numbering) has been substitutedby lysine (K) and in the CH1 domain of the Fab fragment capable ofspecific binding to CD28 the amino acids at position 147 (EU numbering)and at position 213 (EU numbering) have been substituted by glutamicacid (E).

Polynucleotides

The invention further provides isolated polynucleotides encoding abispecific agonistic CD28 antigen binding molecule as described hereinor a fragment thereof. The one or more isolated polynucleotides encodingthe bispecific agonistic CD28 antigen binding molecule of the inventionmay be expressed as a single polynucleotide that encodes the entireantigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional antigen bindingmolecule. For example, the light chain portion of an immunoglobulin maybe encoded by a separate polynucleotide from the heavy chain portion ofthe immunoglobulin. When co-expressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the immunoglobulin.In some aspects, the isolated polynucleotide encodes the entirebispecific agonistic CD28 antigen binding molecule according to theinvention as described herein. In other aspects, the isolatedpolynucleotide encodes a polypeptide comprised in the bispecificagonistic CD28 antigen binding molecule according to the invention asdescribed herein. In certain aspects the polynucleotide or nucleic acidis DNA. In other aspects, a polynucleotide of the present invention isRNA, for example, in the form of messenger RNA (mRNA). RNA of thepresent invention may be single stranded or double stranded.

Recombinant Methods

Bispecific agonistic CD28 antigen binding molecules of the invention maybe obtained, for example, by solid-state peptide synthesis (e.g.Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding thebispecific agonistic CD28 antigen binding molecule or polypeptidefragments thereof, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such polynucleotide may be readily isolated and sequenced usingconventional procedures. In one aspect of the invention, a vector,preferably an expression vector, comprising one or more of thepolynucleotides of the invention is provided. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing the coding sequence of the antibody (fragment) alongwith appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Maniatis et al., MOLECULAR CLONING:A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); andAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing Associates and Wiley Interscience, N.Y. (1989). Theexpression vector can be part of a plasmid, virus, or may be a nucleicacid fragment. The expression vector includes an expression cassetteinto which the polynucleotide encoding the antibody or polypeptidefragments thereof (i.e. the coding region) is cloned in operableassociation with a promoter and/or other transcription or translationcontrol elements. As used herein, a “coding region” is a portion ofnucleic acid which consists of codons translated into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is not translated into anamino acid, it may be considered to be part of a coding region, ifpresent, but any flanking sequences, for example promoters, ribosomebinding sites, transcriptional terminators, introns, 5′ and 3′untranslated regions, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g. on a single vector, or in separate polynucleotideconstructs, e.g. on separate (different) vectors. Furthermore, anyvector may contain a single coding region, or may comprise two or morecoding regions, e.g. a vector of the present invention may encode one ormore polypeptides, which are post- or co-translationally separated intothe final proteins via proteolytic cleavage. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a polynucleotide encoding theantibody of the invention or polypeptide fragments thereof, or variantsor derivatives thereof. Heterologous coding regions include withoutlimitation specialized elements or motifs, such as a secretory signalpeptide or a heterologous functional domain. An operable association iswhen a coding region for a gene product, e.g. a polypeptide, isassociated with one or more regulatory sequences in such a way as toplace expression of the gene product under the influence or control ofthe regulatory sequence(s). Two DNA fragments (such as a polypeptidecoding region and a promoter associated therewith) are “operablyassociated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription.

Suitable promoters and other transcription control regions are disclosedherein. A variety of transcription control regions are known to thoseskilled in the art. These include, without limitation, transcriptioncontrol regions, which function in vertebrate cells, such as, but notlimited to, promoter and enhancer segments from cytomegaloviruses (e.g.the immediate early promoter, in conjunction with intron-A), simianvirus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Roussarcoma virus). Other transcription control regions include thosederived from vertebrate genes such as actin, heat shock protein, bovinegrowth hormone and rabbit â-globin, as well as other sequences capableof controlling gene expression in eukaryotic cells. Additional suitabletranscription control regions include tissue-specific promoters andenhancers as well as inducible promoters (e.g. promoters inducibletetracyclins). Similarly, a variety of translation control elements areknown to those of ordinary skill in the art. These include, but are notlimited to ribosome binding sites, translation initiation andtermination codons, and elements derived from viral systems(particularly an internal ribosome entry site, or IRES, also referred toas a CITE sequence). The expression cassette may also include otherfeatures such as an origin of replication, and/or chromosome integrationelements such as retroviral long terminal repeats (LTRs), oradeno-associated viral (AAV) inverted terminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the antibody or polypeptide fragments thereof is desired, DNAencoding a signal sequence may be placed upstream of the nucleic acid anantibody of the invention or polypeptide fragments thereof. According tothe signal hypothesis, proteins secreted by mammalian cells have asignal peptide or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Those of ordinary skill in theart are aware that polypeptides secreted by vertebrate cells generallyhave a signal peptide fused to the N-terminus of the polypeptide, whichis cleaved from the translated polypeptide to produce a secreted or“mature” form of the polypeptide. In certain embodiments, the nativesignal peptide, e.g. an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, may be used. Forexample, the wild-type leader sequence may be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling thebispecific agonistic CD28 antigen binding molecule may be includedwithin or at the ends of the polynucleotide encoding an antibody of theinvention or polypeptide fragments thereof.

In a further aspect of the invention, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one aspect, a host cellcomprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) an antibody of theinvention of the invention. As used herein, the term “host cell” refersto any kind of cellular system which can be engineered to generate thefusion proteins of the invention or fragments thereof. Host cellssuitable for replicating and for supporting expression of antigenbinding molecules are well known in the art. Such cells may betransfected or transduced as appropriate with the particular expressionvector and large quantities of vector containing cells can be grown forseeding large scale fermenters to obtain sufficient quantities of theantigen binding molecule for clinical applications. Suitable host cellsinclude prokaryotic microorganisms, such as E. coli, or variouseukaryotic cells, such as Chinese hamster ovary cells (CHO), insectcells, or the like. For example, polypeptides may be produced inbacteria in particular when glycosylation is not needed. Afterexpression, the polypeptide may be isolated from the bacterial cellpaste in a soluble fraction and can be further purified. In addition toprokaryotes, eukaryotic microbes such as filamentous fungi or yeast aresuitable cloning or expression hosts for polypeptide-encoding vectors,including fungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006).

Suitable host cells for the expression of (glycosylated) polypeptidesare also derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants). Vertebrate cells may also beused as hosts. For example, mammalian cell lines that are adapted togrow in suspension may be useful. Other examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);human embryonic kidney line (293 or 293T cells as described, e.g., inGraham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells(BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather,Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), Africangreen monkey kidney cells (VERO-76), human cervical carcinoma cells(HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A),human lung cells (W138), human liver cells (Hep G2), mouse mammary tumorcells (MMT 060562), TRI cells (as described, e.g., in Mather et al.,Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad SciUSA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 andSp2/0. For a review of certain mammalian host cell lines suitable forprotein production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells, e.g., mammaliancultured cells, yeast cells, insect cells, bacterial cells and plantcells, to name only a few, but also cells comprised within a transgenicanimal, transgenic plant or cultured plant or animal tissue. In oneembodiment, the host cell is a eukaryotic cell, preferably a mammaliancell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonickidney (HEK) cell or a lymphoid cell (e.g., YO, NS0, Sp20 cell).Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an immunoglobulin, may be engineered so asto also express the other of the immunoglobulin chains such that theexpressed product is an immunoglobulin that has both a heavy and a lightchain.

In one aspect, a method of producing a bispecific agonistic CD28 antigenbinding molecule of the invention or polypeptide fragments thereof isprovided, wherein the method comprises culturing a host cell comprisingpolynucleotides encoding the antibody of the invention or polypeptidefragments thereof, as provided herein, under conditions suitable forexpression of the antibody of the invention or polypeptide fragmentsthereof, and recovering the antibody of the invention or polypeptidefragments thereof from the host cell (or host cell culture medium).

In certain aspects the moieties capable of specific binding to a targetcell antigen (e.g. Fab fragments) forming part of the antigen bindingmolecule comprise at least an immunoglobulin variable region capable ofbinding to an antigen. Variable regions can form part of and be derivedfrom naturally or non-naturally occurring antibodies and fragmentsthereof. Methods to produce polyclonal antibodies and monoclonalantibodies are well known in the art (see e.g. Harlow and Lane,“Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory, 1988).Non-naturally occurring antibodies can be constructed using solidphase-peptide synthesis, can be produced recombinantly (e.g. asdescribed in U.S. Pat. No. 4,186,567) or can be obtained, for example,by screening combinatorial libraries comprising variable heavy chainsand variable light chains (see e.g. U.S. Pat. No. 5,969,108 toMcCafferty).

Any animal species of immunoglobulin can be used in the invention.Non-limiting immunoglobulins useful in the present invention can be ofmurine, primate, or human origin. If the fusion protein is intended forhuman use, a chimeric form of immunoglobulin may be used wherein theconstant regions of the immunoglobulin are from a human. A humanized orfully human form of the immunoglobulin can also be prepared inaccordance with methods well known in the art (see e.g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855(1984); Morrison and Oi, Adv Immunol 44, (1988); Verhoeyen et al.,Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217(1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimkaet al., Br J Cancer 83, 252-260 (2000) (describing the “guidedselection” approach to FR shuffling). Particular immunoglobulinsaccording to the invention are human immunoglobulins. Human antibodiesand human variable regions can be produced using various techniquesknown in the art. Human antibodies are described generally in van Dijkand van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg,Curr Opin Immunol 20, 450-459 (2008). Human variable regions can formpart of and be derived from human monoclonal antibodies made by thehybridoma method (see e.g. Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Humanantibodies and human variable regions may also be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge (see e.g. Lonberg,Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variableregions may also be generated by isolating Fv clone variable regionsequences selected from human-derived phage display libraries (see e.g.,Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien etal., ed., Human Press, Totowa, N J, 2001); and McCafferty et al., Nature348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv (scFv)fragments or as Fab fragments.

In certain aspects, the bispecific agonistic CD28 antigen bindingmolecules are engineered to have enhanced binding affinity according to,for example, the methods disclosed in PCT publication WO 2012/020006(see Examples relating to affinity maturation) or U.S. Pat. Appl. Publ.No. 2004/0132066. The ability of the antigen binding molecules of theinvention to bind to a specific antigenic determinant can be measuredeither through an enzyme-linked immunosorbent assay (ELISA) or othertechniques familiar to one of skill in the art, e.g. surface plasmonresonance technique (Liljeblad, et al., Glyco J 17, 323-329 (2000)), andtraditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).Competition assays may be used to identify an antigen binding moleculethat competes with a reference antibody for binding to a particularantigen. In certain embodiments, such a competing antigen bindingmolecule binds to the same epitope (e.g. a linear or a conformationalepitope) that is bound by the reference antigen binding molecule.Detailed exemplary methods for mapping an epitope to which an antigenbinding molecule binds are provided in Morris (1996) “Epitope MappingProtocols”, in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, NJ). In an exemplary competition assay, immobilized antigen isincubated in a solution comprising a first labeled antigen bindingmolecule that binds to the antigen and a second unlabeled antigenbinding molecule that is being tested for its ability to compete withthe first antigen binding molecule for binding to the antigen. Thesecond antigen binding molecule may be present in a hybridomasupernatant. As a control, immobilized antigen is incubated in asolution comprising the first labeled antigen binding molecule but notthe second unlabeled antigen binding molecule. After incubation underconditions permissive for binding of the first antibody to the antigen,excess unbound antibody is removed, and the amount of label associatedwith immobilized antigen is measured. If the amount of label associatedwith immobilized antigen is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantigen binding molecule is competing with the first antigen bindingmolecule for binding to the antigen. See Harlow and Lane (1988)Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, NY).

Bispecific agonistic CD28 antigen binding molecules of the inventionprepared as described herein may be purified by art-known techniquessuch as high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, affinity chromatography, sizeexclusion chromatography, and the like. The actual conditions used topurify a particular protein will depend, in part, on factors such as netcharge, hydrophobicity, hydrophilicity etc., and will be apparent tothose having skill in the art. For affinity chromatography purificationan antibody, ligand, receptor or antigen can be used to which theantigen binding molecule binds. For example, for affinity chromatographypurification of antigen binding molecules of the invention, a matrixwith protein A or protein G may be used. Sequential Protein A or Gaffinity chromatography and size exclusion chromatography can be used toisolate an antigen binding molecule essentially as described in theExamples. The purity of the CD28 antigen binding molecule or fragmentsthereof can be determined by any of a variety of well-known analyticalmethods including gel electrophoresis, high pressure liquidchromatography, and the like. For example, the CD28 antigen bindingmolecule expressed as described in the Examples were shown to be intactand properly assembled as demonstrated by reducing and non-reducingSDS-PAGE.

Assays

The bispecific agonistic CD28 antigen binding molecules provided hereinmay be identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

1. Affinity Assays

The affinity of the antigen binding molecule provided herein for thecorresponding target can be determined in accordance with the methodsset forth in the Examples by surface plasmon resonance (SPR), usingstandard instrumentation such as a Proteon instrument (Bio-rad), andreceptors or target proteins such as may be obtained by recombinantexpression. The affinity of the TNF family ligand trimer-containingantigen binding molecule for the target cell antigen can also bedetermined by surface plasmon resonance (SPR), using standardinstrumentation such as a Proteon instrument (Bio-rad), and receptors ortarget proteins such as may be obtained by recombinant expression. Aspecific illustrative and exemplary embodiment for measuring bindingaffinity is described in Example 4. According to one aspect, K_(D) ismeasured by surface plasmon resonance using a Proteon® machine (Bio-Rad)at 25° C.

2. Binding Assays and Other Assays

Binding of the bispecific antigen binding molecule provided herein tothe corresponding receptor expressing cells may be evaluated using celllines expressing the particular receptor or target antigen, for exampleby flow cytometry (FACS). In one aspect, CHO cells expressing human CD28(parental cell line CHO-k1 ATCC #CCL-61, modified to stably overexpresshuman CD28) are used in the binding assay.

In a further aspect, cancer cell lines expressing the target cellantigen, for example FAP or CEA, CD19 or CD79b, were used to demonstratethe binding of the bispecific antigen binding molecules to the targetcell antigen.

3. Activity Assays

In one aspect, assays are provided for identifying CD28 antigen bindingmolecules having biological activity. Biological activity may include,e.g. T cell proliferation and cytokine secretion as measured with themethod as described in Example 6 or tumor cell killing as measured inExample 7. Antibodies having such biological activity in vivo and/or invitro are also provided.

Pharmaceutical Compositions, Formulations and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the bispecific agonistic CD28 antigen bindingmolecules provided herein, e.g., for use in any of the below therapeuticmethods. In one embodiment, a pharmaceutical composition comprises abispecific agonistic CD28 antigen binding molecule provided herein andat least one pharmaceutically acceptable excipient. In another aspect, apharmaceutical composition comprises a bispecific agonistic CD28 antigenbinding molecule provided herein and at least one additional therapeuticagent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more bispecific antigenbinding molecules dissolved or dispersed in a pharmaceuticallyacceptable excipient. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that aregenerally non-toxic to recipients at the dosages and concentrationsemployed, i.e. do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one bispecific agonistic CD28 antigen binding moleculeand optionally an additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. In particular, the compositionsare lyophilized formulations or aqueous solutions. As used herein,“pharmaceutically acceptable excipient” includes any and all solvents,buffers, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g. antibacterial agents, antifungal agents), isotonicagents, salts, stabilizers and combinations thereof, as would be knownto one of ordinary skill in the art.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the TNF family ligand trimer-containing antigen bindingmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the bispecific agonistic CD28antigen binding molecule may be in powder form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the fusion proteinsof the invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated below, as required.Sterility may be readily accomplished, e.g., by filtration throughsterile filtration membranes. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The composition must be stable under theconditions of manufacture and storage, and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Itwill be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Suitable pharmaceutically acceptable excipients include, but are notlimited to: buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof. Exemplarypharmaceutically acceptable excipients herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer. In addition to the compositionsdescribed previously, the bispecific agonistic CD28 antigen bindingmolecule may also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the bispecific agonistic CD28 antigen binding molecule maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

Pharmaceutical compositions comprising the bispecific agonistic CD28antigen binding molecule of the invention may be manufactured by meansof conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. The bispecific agonistic CD28 antigen bindingmolecule of the invention may be formulated into a composition in a freeacid or base, neutral or salt form. Pharmaceutically acceptable saltsare salts that substantially retain the biological activity of the freeacid or base. These include the acid addition salts, e.g. those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms. The composition herein may alsocontain more than one active ingredients as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. Such active ingredients aresuitably present in combination in amounts that are effective for thepurpose intended. The formulations to be used for in vivo administrationare generally sterile. Sterility may be readily accomplished, e.g., byfiltration through sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the bispecific agonistic CD28 antigen binding molecules providedherein may be used in therapeutic methods, either alone or incombination.

In one aspect, a bispecific agonistic CD28 antigen binding molecule foruse as a medicament is provided. In further aspects, a bispecificagonistic CD28 antigen binding molecule for use in treating cancer isprovided. In certain aspects, a bispecific agonistic CD28 antigenbinding molecule for use in a method of treatment is provided. Incertain aspects, herein is provided a bispecific agonistic CD28 antigenbinding molecule for use in a method of treating an individual havingcancer comprising administering to the individual an effective amount ofthe bispecific agonistic CD28 antigen binding molecule. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent.

In one aspect, the bispecific agonistic CD28 antigen binding moleculefor use in treating a B-cell proliferative disorder. In particularaspects, the bispecific agonistic CD28 antigen binding molecule is foruse in treating a B-cell proliferative disorder selected from the groupconsisting of Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia(ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma(DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginalzone lymphoma (MZL), Multiple myeloma (MM) and Hodgkin lymphoma (HL). Inone particular aspect, the B-cell cancer is non-Hodgkin lymphoma oracute lymphoblastic leukemia. In certain aspects, a bispecific agonisticCD28 antigen binding molecule for use in a method of treatment isprovided. In certain aspects, herein is provided a bispecific agonisticCD28 antigen binding molecule for use in a method of treating anindividual having cancer comprising administering to the individual aneffective amount of the bispecific agonistic CD28 antigen bindingmolecule. In another aspect, provided is a bispecific agonistic CD28antigen binding molecule for use in a method of treating an individualhaving B-cell proliferative disorder, in particular a B-cellproliferative disorder selected from the group consisting of Non-Hodgkinlymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocyticleukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicularlymphoma (FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL),Multiple myeloma (MM) and Hodgkin lymphoma (HL), comprisingadministering to the individual an effective amount of the bispecificagonistic CD28 antigen binding molecule. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent.

In further aspects, a bispecific agonistic CD28 antigen binding moleculeas described herein for use in cancer immunotherapy is provided. Incertain embodiments, a bispecific agonistic CD28 antigen bindingmolecule for use in a method of cancer immunotherapy is provided. An“individual” according to any of the above aspects is preferably ahuman.

In a further aspect, herein is provided for the use of a bispecificagonistic CD28 antigen binding molecule as described herein in themanufacture or preparation of a medicament. In one embodiment, themedicament is for treatment of cancer. In a further aspect, themedicament is for use in a method of treating cancer comprisingadministering to an individual having cancer an effective amount of themedicament. In one such aspect, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. In anotheraspect, the medicament is for treatment of a B-cell proliferativedisorder. In a further aspect, the medicament is for use in a method oftreating cancer or a B-cell proliferative disorder comprisingadministering to an individual having cancer an effective amount of themedicament.

In a further aspect, herein is provided a method for treating a cancer.In one aspect, the method comprises administering to an individualhaving cancer an effective amount of a bispecific agonistic CD28 antigenbinding molecule. In one such aspect, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, as described below. An “individual”according to any of the above aspects may be a human.

In a further aspect, herein are provided pharmaceutical formulationscomprising any of the bispecific agonistic CD28 antigen bindingmolecules as reported herein, e.g., for use in any of the abovetherapeutic methods. In one aspect, a pharmaceutical formulationcomprises any of the bispecific agonistic CD28 antigen binding moleculesas reported herein and a pharmaceutically acceptable carrier. In anotheraspect, a pharmaceutical formulation comprises any of the bispecificagonistic CD28 antigen binding molecules as reported herein and at leastone additional therapeutic agent.

Bispecific agonistic CD28 antigen binding molecules as reported hereincan be used either alone or in combination with other agents in atherapy. For instance, a bispecific agonistic CD28 antigen bindingmolecule as reported herein may be co-administered with at least oneadditional therapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody as reported herein can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one aspect, administration of thebispecific agonistic CD28 antigen binding molecule and administration ofan additional therapeutic agent occur within about one month, or withinabout one, two or three weeks, or within about one, two, three, four,five, or six days, of each other.

An antigen binding molecule as reported herein (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Bispecific agonistic CD28 antigen binding molecules as described hereinwould be formulated, dosed, and administered in a fashion consistentwith good medical practice. Factors for consideration in this contextinclude the particular disorder being treated, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The bispecific agonistic CD28 antigenbinding molecule need not be, but is optionally formulated with one ormore agents currently used to prevent or treat the disorder in question.The effective amount of such other agents depends on the amount ofantibody present in the formulation, the type of disorder or treatment,and other factors discussed above. These are generally used in the samedosages and with administration routes as described herein, or aboutfrom 1 to 99% of the dosages described herein, or in any dosage and byany route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of abispecific agonistic CD28 antigen binding molecule as described herein(when used alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the type of antibody, the severity and course of the disease, whetherthe antibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The bispecificagonistic CD28 antigen binding molecule is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5mg/kg-10 mg/kg) of bispecific agonistic CD28 antigen binding moleculecan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. One typical daily dosage might range from about 1μg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment would generally be sustained until adesired suppression of disease symptoms occurs. One exemplary dosage ofthe antibody would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

Other Agents and Treatments

The bispecific agonistic CD28 antigen binding molecules of the inventionmay be administered in combination with one or more other agents intherapy. For instance, an antigen binding molecule of the invention maybe co-administered with at least one additional therapeutic agent. Theterm “therapeutic agent” encompasses any agent that can be administeredfor treating a symptom or disease in an individual in need of suchtreatment. Such additional therapeutic agent may comprise any activeingredients suitable for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. In certain embodiments, an additional therapeuticagent is another anti-cancer agent, for example a microtubule disruptor,an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, analkylating agent, a hormonal therapy, a kinase inhibitor, a receptorantagonist, an activator of tumor cell apoptosis, or an antiangiogenicagent. In certain aspects, an additional therapeutic agent is animmunomodulatory agent, a cytostatic agent, an inhibitor of celladhesion, a cytotoxic or cytostatic agent, an activator of cellapoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers.

Thus, provided are bispecific agonistic CD28 antigen binding moleculesof the invention or pharmaceutical compositions comprising them for usein the treatment of cancer, wherein the bispecific antigen bindingmolecule is administered in combination with a chemotherapeutic agent,radiation and/or other agents for use in cancer immunotherapy.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The bispecificantigen binding molecule or antibody of the invention are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate. Such combination therapies noted above encompass combinedadministration (where two or more therapeutic agents are included in thesame or separate compositions), and separate administration, in whichcase, administration of the bispecific antigen binding molecule orantibody of the invention can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent and/oradjuvant.

In a further aspect, provided is the bispecific agonistic CD28 antigenbinding molecule as described herein before for use in the treatment ofcancer, wherein the bispecific antigen binding molecule is administeredin combination with another immunomodulator. The term “immunomodulator”refers to any substance including a monoclonal antibody that effects theimmune system. The molecules of the inventions can be consideredimmunomodulators. Immunomodulators can be used as anti-neoplastic agentsfor the treatment of cancer. In one aspect, immunomodulators include,but are not limited to anti-CTLA4 antibodies (e.g. ipilimumab), anti-PD1antibodies (e.g. nivolumab or pembrolizumab), PD-L1 antibodies (e.g.atezolizumab, avelumab or durvalumab), OX-40 antibodies, 4-1BBantibodies and GITR antibodies. Such combination therapies noted aboveencompass combined administration (where two or more therapeutic agentsare included in the same or separate compositions), and separateadministration, in which case, administration of the bispecific antigenbinding molecule can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.

Combination with T Cell Bispecific Antibodies

In one aspect, the bispecific agonistic CD28 antigen binding moleculesof the invention may be administered in combination with T-cellactivating anti-CD3 bispecific antibodies. In one aspect, the T-cellactivating anti-CD3 bispecific antibody specific for a tumor-associatedantigen is an anti-CEA/anti-CD3 bispecific antibody or ananti-MCSP/anti-CD3 bispecific antibody. In one particular aspect, theT-cell activating anti-CD3 bispecific antibody specific for atumor-associated antigen is an anti-CEA/anti-CD3 bispecific antibody.

In one aspect, a bispecific agonistic CD28 antigen binding moleculecomprising at least one antigen binding domain capable of specificbinding to a tumor-associated antigen (TAA) selected from the groupconsisting of Fibroblast Activation Protein (FAP), CarcinoembryonicAntigen (CEA), Folate receptor alpha (FolR1), Melanoma-associatedChondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth FactorReceptor (EGFR), human epidermal growth factor receptor 2 (HER2) andp95HER2 is suitable for administration in combination with ananti-CEA/anti-CD3 bispecific antibody. In another particular aspect, TAAis selected from the group consisting of Fibroblast Activation Protein(FAP), Carcinoembryonic Antigen (CEA), EpCAM, HER3, CD30 or TPBG (5T4).

In a particular aspect, the anti-CD3 bispecific antibody for use in thecombination comprises a first antigen binding domain comprising a heavychain variable region (V_(H)CD3) comprising CDR-H1 sequence of SEQ IDNO:439, CDR-H2 sequence of SEQ ID NO:440, and CDR-H3 sequence of SEQ IDNO:441; and/or a light chain variable region (V_(L)CD3) comprisingCDR-L1 sequence of SEQ ID NO:442, CDR-L2 sequence of SEQ ID NO:443, andCDR-L3 sequence of SEQ ID NO:444. More particularly, the anti-CD3bispecific comprises a first antigen binding domain comprising a heavychain variable region (V_(H)CD3) that is at least 90%, 95%, 96%, 97%,98%, or 99% identical to the amino acid sequence of SEQ ID NO:445 and/ora light chain variable region (V_(L)CD3) that is at least 90%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:446.In a further aspect, the anti-CD3 bispecific antibody comprises a heavychain variable region (V_(H)CD3) comprising the amino acid sequence ofSEQ ID NO:445 and/or a light chain variable region (V_(L)CD3) comprisingthe amino acid sequence of SEQ ID NO:446.

In another aspect, the anti-CD3 bispecific antibody for use in thecombination comprises a first antigen binding domain comprising a heavychain variable region (V_(H)CD3) comprising CDR-H1 sequence of SEQ IDNO:596, CDR-H2 sequence of SEQ ID NO:597, and CDR-H3 sequence of SEQ IDNO:598; and/or a light chain variable region (V_(L)CD3) comprisingCDR-L1 sequence of SEQ ID NO:599, CDR-L2 sequence of SEQ ID NO:600, andCDR-L3 sequence of SEQ ID NO:601. More particularly, the anti-CD3bispecific comprises a first antigen binding domain comprising a heavychain variable region (V_(H)CD3) that is at least 90%, 95%, 96%, 97%,98%, or 99% identical to the amino acid sequence of SEQ ID NO:602 and/ora light chain variable region (V_(L)CD3) that is at least 90%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:603.In a further aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a heavy chain variable region (V_(H)CD3) comprising the aminoacid sequence of SEQ ID NO:602 and/or a light chain variable region(V_(L)CD3) comprising the amino acid sequence of SEQ ID NO:603.

In another aspect, the anti-CD3 bispecific antibody for use in thecombination comprises a first antigen binding domain comprising a heavychain variable region (V_(H)CD3) comprising CDR-H1 sequence of SEQ IDNO:604, CDR-H2 sequence of SEQ ID NO:605, and CDR-H3 sequence of SEQ IDNO:606; and/or a light chain variable region (V_(L)CD3) comprisingCDR-L1 sequence of SEQ ID NO:607, CDR-L2 sequence of SEQ ID NO:608, andCDR-L3 sequence of SEQ ID NO:609. More particularly, the anti-CD3bispecific comprises a first antigen binding domain comprising a heavychain variable region (V_(H)CD3) that is at least 90%, 95%, 96%, 97%,98%, or 99% identical to the amino acid sequence of SEQ ID NO:610 and/ora light chain variable region (V_(L)CD3) that is at least 90%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:611.In a further aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a heavy chain variable region (V_(H)CD3) comprising the aminoacid sequence of SEQ ID NO:610 and/or a light chain variable region(V_(L)CD3) comprising the amino acid sequence of SEQ ID NO:611.

In one particular aspect, the anti-CEA/anti-CD3 bispecific antibodycomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 161, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:162, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 163, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 164.In a further particular embodiment, the bispecific antibody comprises apolypeptide sequence of SEQ ID NO: 161, a polypeptide sequence of SEQ IDNO: 162, a polypeptide sequence of SEQ ID NO: 163 and a polypeptidesequence of SEQ ID NO: 164 (CEA CD3 TCB).

In another particular aspect, the anti-CEA/anti-CD3 bispecific antibodycomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO:165, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO:166, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO:167, and a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO:168. In a further particular embodiment, the bispecific antibodycomprises a polypeptide sequence of SEQ ID NO:165, a polypeptidesequence of SEQ ID NO:166, a polypeptide sequence of SEQ ID NO:167 and apolypeptide sequence of SEQ ID NO:168 (CEACAM5 CD3 TCB).

Particular bispecific antibodies are further described in PCTpublication no. WO 2014/131712 A1. In a further aspect, theanti-CEA/anti-CD3 bispecific antibody may also comprise a bispecific Tcell engager (BiTE®). In a further aspect, the anti-CEA/anti-CD3bispecific antibody is a bispecific antibody as described in WO2007/071426 or WO 2014/131712.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeof the invention comprising an antigen binding domain capable ofspecific binding to a B cell surface antigen may be administered incombination with T-cell activating anti-CD3 bispecific antibodies. Inone aspect, the T-cell activating anti-CD3 bispecific antibody isspecific for a B cell surface antigen, in particular it is ananti-CD20/anti-CD3 bispecific antibody.

The anti-CD20/anti-CD3 bispecific antibodies as used herein arebispecific antibodies comprising a first antigen binding domain thatbinds to CD3, and a second antigen binding domain that binds to CD20.Thus, the anti-CD20/anti-CD3 bispecific antibody as used hereincomprises a first antigen binding domain comprising a heavy chainvariable region (V_(H)CD3) and a light chain variable region (V_(L)CD3),and a second antigen binding domain comprising a heavy chain variableregion (V_(H)CD20) and a light chain variable region (V_(L)CD20).

In a particular aspect, the anti-CD20/anti-CD3 bispecific antibody foruse in the combination comprises a first antigen binding domaincomprising a heavy chain variable region (V_(H)CD3) comprising CDR-H1sequence of SEQ ID NO:439, CDR-H2 sequence of SEQ ID NO:440, and CDR-H3sequence of SEQ ID NO:441; and/or a light chain variable region(V_(L)CD3) comprising CDR-L1 sequence of SEQ ID NO:442, CDR-L2 sequenceof SEQ ID NO:443, and CDR-L3 sequence of SEQ ID NO:444. Moreparticularly, the anti-CD20/anti-CD3 bispecific comprises a firstantigen binding domain comprising a heavy chain variable region(V_(H)CD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO:445 and/or a light chain variableregion (V_(L)CD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO:446. In a furtheraspect, the anti-CD20/anti-CD3 bispecific antibody comprises a heavychain variable region (V_(H)CD3) comprising the amino acid sequence ofSEQ ID NO:445 and/or a light chain variable region (V_(L)CD3) comprisingthe amino acid sequence of SEQ ID NO:446.

In one aspect, the antibody that specifically binds to CD3 is afull-length antibody. In one aspect, the antibody that specificallybinds to CD3 is an antibody of the human IgG class, particularly anantibody of the human IgG1 class. In one aspect, the antibody thatspecifically binds to CD3 is an antibody fragment, particularly a Fabmolecule or a scFv molecule, more particularly a Fab molecule. In aparticular aspect, the antibody that specifically binds to CD3 is acrossover Fab molecule wherein the variable domains or the constantdomains of the Fab heavy and light chain are exchanged (i.e. replaced byeach other). In one aspect, the antibody that specifically binds to CD3is a humanized antibody.

In another aspect, the anti-CD20/anti-CD3 bispecific antibody comprisesa second antigen binding domain comprising a heavy chain variable region(V_(H)CD20) comprising CDR-H1 sequence of SEQ ID NO:447, CDR-H2 sequenceof SEQ ID NO:448, and CDR-H3 sequence of SEQ ID NO:449, and/or a lightchain variable region (V_(L)CD20) comprising CDR-L1 sequence of SEQ IDNO:450, CDR-L2 sequence of SEQ ID NO:451, and CDR-L3 sequence of SEQ IDNO:452. More particularly, the anti-CD20/anti-CD3 bispecific comprises asecond antigen binding domain comprising a heavy chain variable region(V_(H)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence of SEQ ID NO:453 and/or a light chainvariable region (V_(L)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or99% identical to the amino acid sequence of SEQ ID NO:454. In a furtheraspect, the anti-CD20/anti-CD3 bispecific comprises a second antigenbinding domain comprising a heavy chain variable region (V_(H)CD20)comprising the amino acid sequence of SEQ ID NO:453 and/or a light chainvariable region (V_(L)CD20) comprising the amino acid sequence of SEQ IDNO:454.

In another particular aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a third antigen binding domain that binds to CD20. Inparticular, the anti-CD20/anti-CD3 bispecific antibody comprises a thirdantigen binding domain comprising a heavy chain variable region(V_(H)CD20) comprising CDR-H1 sequence of SEQ ID NO:447, CDR-H2 sequenceof SEQ ID NO:448, and CDR-H3 sequence of SEQ ID NO:449; and/or a lightchain variable region (V_(L)CD20) comprising CDR-L1 sequence of SEQ IDNO:450, CDR-L2 sequence of SEQ ID NO:451, and CDR-L3 sequence of SEQ IDNO:452. More particularly, the anti-CD20/anti-CD3 bispecific comprises athird antigen binding domain comprising a heavy chain variable region(V_(H)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence of SEQ ID NO:453 and/or a light chainvariable region (V_(L)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or99% identical to the amino acid sequence of SEQ ID NO:454. In a furtheraspect, the anti-CD20/anti-CD3 bispecific comprises a third antigenbinding domain comprising a heavy chain variable region (V_(H)CD20)comprising the amino acid sequence of SEQ ID NO:453 and/or a light chainvariable region (V_(L)CD20) comprising the amino acid sequence of SEQ IDNO:454.

In a further aspect, the anti-CD20/anti-CD3 bispecific antibody isbispecific antibody, wherein the first antigen binding domain is across-Fab molecule wherein the variable domains or the constant domainsof the Fab heavy and light chain are exchanged, and the second andthird, if present, antigen binding domain is a conventional Fabmolecule.

In another aspect, the anti-CD20/anti-CD3 bispecific antibody isbispecific antibody, wherein (i) the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding domain, the first antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and the third antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain, or (ii) the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingdomain, the second antigen binding domain is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first subunit of the Fcdomain, and the third antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the second subunit of the Fcdomain.

The Fab molecules may be fused to the Fc domain or to each otherdirectly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. In one aspect, said peptide linker is(G₄S)₂ (SEQ ID NO:147). Another suitable such linker comprises thesequence (G₄S)₄ (SEQ ID NO:152). Additionally, linkers may comprise (aportion of) an immunoglobulin hinge region. Particularly where a Fabmolecule is fused to the N-terminus of an Fc domain subunit, it may befused via an immunoglobulin hinge region or a portion thereof, with orwithout an additional peptide linker. In a further aspect, theanti-CD20/anti-CD3 bispecific antibody comprises an Fc domain comprisingone or more amino acid substitutions that reduce binding to an Fcreceptor and/or effector function. In particular, the anti-CD20/anti-CD3bispecific antibody comprises an IgG1 Fc domain comprising the aminoacid substitutions L234A, L235A and P329G.

In a particular aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 455, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:456, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 457, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 458.In a further particular embodiment, the bispecific antibody comprises apolypeptide sequence of SEQ ID NO: 455, a polypeptide sequence of SEQ IDNO: 456, a polypeptide sequence of SEQ ID NO: 457 and a polypeptidesequence of SEQ ID NO: 458 (CD20 TCB).

Particular bispecific antibodies are described in PCT publication no. WO2016/020309 A1 or in WO 2015/095392 A1. In a further aspect, theanti-CD20/anti-CD3 bispecific antibody may also comprise a bispecific Tcell engager (BITE®). In a further aspect, the anti-CD20/anti-CD3bispecific antibody is XmAb ° 13676. In another aspect, the bispecificantibody is REGN1979. In another aspect, the bispecific antibody isFBTA05 (Lymphomun).

In another aspect of the invention, the bispecific agonistic CD28antigen binding molecule of the invention is for use in a method fortreating or delaying progression of cancer, wherein the bispecificagonistic CD28 antigen binding molecule is used in combination with ananti-CD20/anti-CD3 bispecific antibody, and additionally they arecombined with an agent blocking PD-L1/PD-1 interaction. An agentblocking PD-L1/PD-1 interaction is a PD-L1 binding antagonist or a PD-1binding antagonist. In particular, the agent blocking PD-L1/PD-1interaction is an anti-PD-L1 antibody or an anti-PD-1 antibody.

In another aspect, a bispecific agonistic CD28 antigen binding moleculeof the invention comprising an antigen binding domain capable ofspecific binding to MM cell surface antigen may be administered incombination with T-cell activating anti-CD3 bispecific antibodies. Inone aspect, the T-cell activating anti-CD3 bispecific antibody isspecific for a MM cell surface antigen, in particular it is ananti-GPRC5D/anti-CD3 bispecific antibody.

In one particular aspect, the anti-GPRC5D/anti-CD3 bispecific antibodycomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 398, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:399, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 404, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 405.In a further particular aspect, the bispecific antibody comprises apolypeptide sequence of SEQ ID NO: 398, a polypeptide sequence of SEQ IDNO: 399, a polypeptide sequence of SEQ ID NO: 404 and a polypeptidesequence of SEQ ID NO: 405 (GPRC5D CD3 TCB).

In another aspect, provided is a combination product comprising abispecific agonistic CD28 antigen binding molecule as described hereinand a T-cell activating anti-CD3 bispecific antibody. In one aspect, theT-cell activating anti-CD3 bispecific antibody specific for atumor-associated antigen is an anti-CEA/anti-CD3 bispecific antibody oran anti-MCSP/anti-CD3 bispecific antibody. In one particular aspect, theT-cell activating anti-CD3 bispecific antibody specific for atumor-associated antigen is an anti-CEA/anti-CD3 bispecific antibody. Inanother aspect, the T-cell activating anti-CD3 bispecific antibodyspecific for a tumor-associated antigen is an anti-CD20/anti-CD3bispecific antibody. In a further aspect, the T-cell activating anti-CD3bispecific antibody specific for a tumor-associated antigen is ananti-GPRC5D/anti-CD3 bispecific antibody.

Combination with Agents Blocking PD-L1/PD-1 Interaction

In one aspect, the bispecific agonistic CD28 antigen binding moleculesof the invention may be administered in combination with agents blockingPD-L1/PD-1 interaction such as a PD-L1 binding antagonist or a PD-1binding antagonist, in particular an anti-PD-L1 antibody or an anti-PD-1antibody.

In one aspect, the agent blocking PD-L1/PD-1 interaction is ananti-PD-L1 antibody. The term “PD-L1”, also known as CD274 or B7-H1,refers to any native PD-L1 from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), in particular to “humanPD-L1”. The amino acid sequence of complete human PD-L1 is shown inUniProt (world wide web uniprot.org) accession no. Q9NZQ7 (SEQ IDNO:459). The term “PD-L1 binding antagonist” refers to a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of PD-L1 with either one ormore of its binding partners, such as PD-1, B7-1. In some aspects, aPD-L1 binding antagonist is a molecule that inhibits the binding ofPD-L1 to its binding partners. In a specific aspect, the PD-L1 bindingantagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In someaspects, the PD-L1 binding antagonists include anti-PD-L1 antibodies,antigen binding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-L1 with one or more of its binding partners, such asPD-1, B7-1. In one aspect, a PD-L1 binding antagonist reduces thenegative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-L1 soas to render a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In particular, a PD-L1binding antagonist is an anti-PD-L1 antibody. The term “anti-PD-L1antibody” or “antibody binding to human PD-L1” or “antibody thatspecifically binds to human PD-L1” or “antagonistic anti-PD-L1” refersto an antibody specifically binding to the human PD-L1 antigen with abinding affinity of KD-value of 1.0×10⁻⁸ mol/1 or lower, in one aspectof a KD-value of 1.0×10⁻⁹ mol/1 or lower. The binding affinity isdetermined with a standard binding assay, such as surface plasmonresonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden). In aparticular aspect, the agent blocking PD-L1/PD-1 interaction is ananti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody isselected from the group consisting of atezolizumab (MPDL3280A, RG7446),durvalumab (MEDI4736), avelumab (MSB0010718C) and MDX-1105. In aspecific aspect, an anti-PD-L1 antibody is YW243.55.570 describedherein. In another specific aspect, an anti-PD-L1 antibody is MDX-1105described herein. In still another specific aspect, an anti-PD-L1antibody is MEDI4736 (durvalumab). In yet a further aspect, ananti-PD-L1 antibody is MSB0010718C (avelumab). More particularly, theagent blocking PD-L1/PD-1 interaction is atezolizumab (MPDL3280A). Inanother aspect, the agent blocking PD-L1/PD-1 interaction is ananti-PD-L1 antibody comprising a heavy chain variable domain VH(PDL-1)of SEQ ID NO:460 and a light chain variable domain VL(PDL-1) of SEQ IDNO:461. In another aspect, the agent blocking PD-L1/PD-1 interaction isan anti-PD-L1 antibody comprising a heavy chain variable domainVH(PDL-1) of SEQ ID NO:462 and a light chain variable domain VL(PDL-1)of SEQ ID NO:463.

The term “PD-1”, also known as CD279, PD1 or programmed cell deathprotein 1, refers to any native PD-L1 from any vertebrate source,including mammals such as primates (e.g. humans) non-human primates(e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), inparticular to the human protein PD-1 with the amino acid sequence asshown in UniProt (world wide web.uniprot.org) accession no. Q15116 (SEQID NO:464). The term “PD-1 binding antagonist” refers to a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In someembodiments, the PD-1 binding antagonist inhibits the binding of PD-1 toPD-L1. In some embodiments, the PD-1 binding antagonist inhibits thebinding of PD-1 to PD-L2. In some embodiments, the PD-1 bindingantagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. Inparticular, a PD-L1 binding antagonist is an anti-PD-L1 antibody. Theterm “anti-PD-1 antibody” or “antibody binding to human PD-1” or“antibody that specifically binds to human PD-1” or “antagonisticanti-PD-1” refers to an antibody specifically binding to the human PD1antigen with a binding affinity of KD-value of 1.0×10⁻⁸ mol/1 or lower,in one aspect of a KD-value of 1.0×10⁻⁹ mol/1 or lower. The bindingaffinity is determined with a standard binding assay, such as surfaceplasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden).In one aspect, the agent blocking PD-L1/PD-1 interaction is an anti-PD-1antibody. In a specific aspect, the anti-PD-1 antibody is selected fromthe group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab),CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, andBGB-108, in particular from pembrolizumab and nivolumab. In anotheraspect, the agent blocking PD-L1/PD-1 interaction is an anti-PD-1antibody comprising a heavy chain variable domain VH(PD-1) of SEQ IDNO:465 and a light chain variable domain VL(PD-1) of SEQ ID NO:466. Inanother aspect, the agent blocking PD-L1/PD-1 interaction is ananti-PD-1 antibody comprising a heavy chain variable domain VH(PD-1) ofSEQ ID NO:467 and a light chain variable domain VL(PD-1) of SEQ IDNO:468.

In another aspect, provided is a combination product comprising abispecific agonistic CD28 antigen binding molecule as described hereinand an agents blocking PD-L1/PD-1 interaction such as a PD-L1 bindingantagonist or a PD-1 binding antagonist, in particular an anti-PD-L1antibody or an anti-PD-1 antibody.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the therapeutic agent can occur prior to,simultaneously, and/or following, administration of an additionaltherapeutic agent or agents. In one embodiment, administration of thetherapeutic agent and administration of an additional therapeutic agentoccur within about one month, or within about one, two or three weeks,or within about one, two, three, four, five, or six days, of each other.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is a bispecific agonistic CD28 antigen bindingmolecule of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice. Moreover,the article of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises abispecific agonistic CD28 antigen binding molecule of the invention; and(b) a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

TABLE B (Sequences): SEQ ID NO: Name Sequence 1 hu CD28UniProt no. P10747, version 1 2 hu FAP UniProt no. Q12884, version 168 3hu CEA UniProt accession no. P06731 4 FAP (28H1) CDR-H1 SHAMS 5FAP (28H1) CDR-H2 AIWASGEQYYADSVKG 6 FAP (28H1) CDR-H3 GWLGNFDY 7FAP (28H1) CDR-L1 RASQSVSRSYLA 8 FAP (28H1) CDR-L2 GASTRAT 9FAP (28H1) CDR-L3 QQGQVIPPT 10 FAP(28H1) VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCAKGWLGNFDYWGQGTLVTVSS11 FAP(28H1) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QGQVIPPTFGQGTKVEIK 12FAP(4B9) CDR-H1 SYAMS 13 FAP(4B9) CDR-H2 AIIGSGASTYYADSVKG 14FAP(4B9) CDR-H3 GWFGGFNY 15 FAP(4B9) CDR-L1 RASQSVTSSYLA 16FAP(4B9) CDR-L2 VGSRRAT 17 FAP(4B9) CDR-L3 QQGIMLPPT 18 FAP(4B9) VHEVQLLESGGGLVQPGGSLRLSCAASGFTESSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 19 FAP(4B9) VLEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QGIMLPPTFGQGTKVEIK 20CD28(SA) CDR-H1 SYYIH 21 CD28(SA) CDR-H2 CIYPGNVNTNYNEKFKD 22CD28(SA) CDR-H3 SHYGLDWNEDV 23 CD28(SA) CDR-L1 HASQNIYVWLN 24CD28(SA) CDR-L2 KASNLHT 25 CD28(SA) CDR-L3 QQGQTYPYT 26 CD28(SA) VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS 27 CD28(SA) VLDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ GQTYPYTFGGGTKVEIK 28CD28(mAb 9.3) CDR- DYGVH H1 29 CD28(mAb 9.3) CDR- VIWAGGGTNYNSALMS H2 30CD28(mAb 9.3) CDR- DKGYSYYYSMDY H3 31 CD28(mAb 9.3) CDR- RASESVEYYVTSLMQL1 32 CD28(mAb 9.3) CDR- AASNVES L2 33 CD28(mAb 9.3) CDR- QQSRKVPYT L334 CD28(mAb 9.3) VH EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSS 35 CD28(mAb 9.3) VLDIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMY FCQQSRKVPYTFGGGTKLEIK 36CD28 CDR-H1 SYYIH consensus 37 CD28 CDR-H2SIYPX₁X₂X₃X₄TNYNEKFKD, wherein consensus X₁ is G or R X₂ is N or DX₃ is V or G X₄ is N or Q or A 38 CD28 CDR-H3 SHYGX₅DX₆NFDV, whereinconsensus X₅ is L or A X₆ is W or H or Y or F 39 CD28 CDR-L1X₇ASQX₈IX₉X₁₀X₁₁LN, wherein consensus X₇ is H or R X₈ is N or GX₉ is Y or S X₁₀ is V or N X₁₁ is W or H or For Y 40 CD28 CDR-L2X₁₂X₁₃SX₁₄LX₁₅X₁₆, wherein consensus X₁₂ is K or Y X₁₃ is A or TX₁₄ is N or S X₁₅ is H or Y X₁₆ is T or S 41 CD28 CDR-L3QQX₁₇QTYPYT, wherein consensus X₁₇ is G or A 42 CD28 VH variant aQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGLDWNFDVWGQGTTVTVSS43 CD28 VH variant b QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGLDHNFDVWGQGTTVTVSS44 CD28 VH variant c QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGADHNFDVWGQGTTVTVSS45 CD28 VH variant d QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPRDGQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGLDYNFDVWGQGTTVTVSS46 CD28 VH variant e QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGLDWNFDVWGQGTTVTVSS47 CD28 VH variant f QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGLDFNFDVWGQGTTVTVSS48 CD28 VH variant g QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPRNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGLDHNFDVWGQGTTVTVSS49 CD28 VH variant h QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPRDVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSH YGLDHNFDVWGQGTTVTVSS50 CD28 VH variant i EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYYIHWVRQAPGKGLEWVASIYPGNVNTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCTRSH YGLDWNFDVWGQGTTVTVSS51 CD28 VH variant j EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYYIHWVRQAPGKGLEWVASIYPGNVATRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCTRSH YGLDWNFDVWGQGTTVTVSS52 CD28 VL variant k DIQMTQSPSSLSASVGDRVTITCHASQNIYVHLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAQTYPYTFGG GTKVEIK 53CD28 VL variant l DIQMTQSPSSLSASVGDRVTITCHASQNIYVFLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 54CD28 VL variant m DIQMTQSPSSLSASVGDRVTITCHASQNIYVYLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 55CD28 VL variant n DIQMTQSPSSLSASVGDRVTITCHASQGISNYLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 56CD28 VL variant o DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 57CD28 VL variant p DIQMTQSPSSLSASVGDRVTITCHASQGISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 58CD28 VL variant q DIQMTQSPSSLSASVGDRVTITCHASQGISNHLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 59CD28 VL variant r DIQMTQSPSSLSASVGDRVTITCHASQGIYVYLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 60CD28 VL variant s DIQMTQSPSSLSASVGDRVTITCHASQGISVYLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGG GTKVEIK 61CD28 VL variant t DIQMTQSPSSLSASVGDRVTITCRASQNIYVWLNWYQQKPGKAPKLLIYKASNLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGQ GTKLEIK 62CD28(SA) light chain DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 63 CD28(SA) hu IgG4QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL heavy chainEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 64 CD28(SA) hu IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL PGLALA heavy chainEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 65 VL-CD28(SA)-DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK CL“RK”LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 66 CD28(SA) hu IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL PGLALA Fc knobEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 67 FAP(4B9) VL-CH huEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAP IgG1 PGLALA Fc holeRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 68 FAP(4B9) VH-CkappaEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 69 CD28(SA) VHCH-QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL VHCH Fc knobEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD FAP(4B9) VHTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSS PGLALAKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGENYW GQGTLVTVSS 70CD28(SA) VHCH- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLVHCH Fc hole EWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD FAP(4B9) VLTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSS PGLALAKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEI K 71 CD28(SA) VHCH- FcQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL knob FAP(4B9) VHEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVS S 72 CD28(SA) VHCH- FcQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hole FAP(4B9) VLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIK 73 CD28(SA) VHCHQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL “EE”- Fc PGLALAEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD FAP(4B9) VHCLTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 74FAP(4B9) VLCH1 EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD 75 CD28(SA) VLCH1-DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK FAP(4B9) VHCH1LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ “EE”- Fc knobGQTYPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALG PGLALACLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 76 FAP(4B9) VHCH1EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL “EE”- Fc hole PGLALAEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 77 CD28(SA) VHCLQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 78 FAP(4B9) VLCL “RK”EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 79 Fc hole PGLALADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 80 Fc knob -FAP(4B9) VHDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVT VSS 81 CD28(SA) VHCH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL “EE”- Fc PGLALAEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD CEA VHCLTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 82CEAVLCH1 QAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 83 CD28(SA) VHCH1- FcQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL knob CEA VHEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTT VTVSS 84CD28(SA) VHCH1- Fc QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLhole CEA VL EWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVL 85 CD28(SA) VHCH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL “EE”- Fc hole PGLALAEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD HYRFTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP 86 Fc knob PGLALADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 87 CEA VL-CH1 hu IgG1QAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSP PGLALA Fc holePQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 88 CEAVH-CLEVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 89 CD28(mAb 9.3) lightDIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPG chainQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 90 CD28(mAb 9.3) huEVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL IgG1 PGLALA heavyEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT chainAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 91 CD28(mAb 9.3) hu IgGEVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL light chain “RK”EWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 CD28(mAb 9.3) huDIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPG IgG1 PGLALA Fc knobQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMY “EE”FCQQSRKVPYTFGGGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 93 CD28(mAb 9.3)EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL VHCH-VHCH Fc knobEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT FAP(4B9) VHAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSS PGLALAKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGENYW GQGTLVTVSS 94CD28(mAb 9.3) EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLVHCH-VHCH Fc hole EWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTFAP(4B9) VL AVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSS PGLALAKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVELKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEI K 95 CD28(mAb 9.3)EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL VHCH- Fc knobEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT FAP(4B9) VHAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVS S 96 CD28(mAb 9.3)EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL VHCH- Fc holeEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT FAP(4B9) VLAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIK 97 CD28(mAb 9.3) VHCHEVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL “EE”- Fc PGLALAEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT FAP(4B9) VHCLAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 98CD28(mAb 9.3) VLCL DIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPG “RK”QPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 99 FAP(4B9) VLCH1EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 100 CD28(mAb 9.3)DIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPG VLCH1- FAP(4B9)QPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMY VHCHI“EE”- Fc knobFCQQSRKVPYTFGGGTKLEIKSSASTKGPSVFPLAPSSKSTSGGT PGLALAAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 101 CD28(mAb 9.3) VHCLEVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 102 CD28(mAb 9.3)EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL VHCH1 “EE”- FcEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT PGLALA CEA VHCLAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 103CD28(mAb 9.3) EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLVHCH1- Fc knob CEA EWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT VHAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTT VTVSS 104 CD28(mAb 9.3)EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL VHCH1- Fc hole CEAEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT VLAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVL 105 CD28(mAb 9.3)EVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGL VHCH1 “EE”- Fc holeEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDT PGLALA HYRFAVYYCARDKGYSYYYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 106 CD28(mAb 9.3) VLCLDIELTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPG “RK”QPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 107 CD28(SA) VHCH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL “EE” Fc hole PGLALAEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD FAP(4B9) VH -TAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSS CEA(Medi-565) VHCLKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC108 CD28(SA) VHCH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL“EE” Fc knob PGLALA EWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDFAP(4B9) VL TAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIK 109 CEA VLCH1QAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 110 CD28(SA) VHCH1 FcQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hole PGLALAEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD FAP(4B9) VH - CEATAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSS VHKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQG TTVTVSS 111CD28(SA) VHCH1 Fc QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLknob PGLALA EWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDFAP(4B9) VL - CEA TAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSS VLKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVL 112 VH (CD28 SA) CH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL (EE)- Fc knobEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD PGLALATAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 113 VH (CD28 variant g)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL CH1 (EE) - Fc knobEWIGSIYPRNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD PGLALATAVYFCTRSHYGLDHNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 114 VH (CD28 variant f)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL CH1 (EE) - Fc knobEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD PGLALATAVYFCTRSHYGLDFNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 115 VH (CD28 variant j)EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYYIHWVRQAPGKGL CH1 (EE) - Fc knobEWVASIYPGNVATRYADSVKGRFTISADTSKNTAYLQMNSLRAED PGLALATAVYYCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 116 VH (CD28 variant e)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL CH1 (EE)- Fc knobEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD PGLALATAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 117 VH (CD28 variant b)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL CH1 (EE) - Fc knobEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD PGLALATAVYFCTRSHYGLDHNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 118 VH (CD28 variant a)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL CH1 (EE) - Fc knobEWIGSIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD PGLALATAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 119 VH (CD28 variant i)EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYYIHWVRQAPGKGL CH1 (EE) - Fc knobEWVASIYPGNVNTRYADSVKGRFTISADTSKNTAYLQMNSLRAED PGLALATAVYYCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 120 VL (CD28 variant k)-DIQMTQSPSSLSASVGDRVTITCHASQNIYVHLNWYQQKPGKAPK CL (RK)LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 121 VL (CD28 variant 1)-CLDIQMTQSPSSLSASVGDRVTITCHASQNIYVFLNWYQQKPGKAPK (RK)LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 122 VL (CD28 variant m)-DIQMTQSPSSLSASVGDRVTITCHASQNIYVYLNWYQQKPGKAPK CL (RK)LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 123 VL (CD28 variant r)-CLDIQMTQSPSSLSASVGDRVTITCHASQGIYVYLNWYQQKPGKAPK (RK)LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 124 VL (CD28 variant s)-DIQMTQSPSSLSASVGDRVTITCHASQGISVYLNWYQQKPGKAPK CL (RK)LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 125 VL (CD28 variant t)-CLDIQMTQSPSSLSASVGDRVTITCRASQNIYVWLNWYQQKPGKAPK (RK)LLIYKASNLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 126 Fc hole PGLALA,DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD HYRFVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP 127 CEA CDR-H1 SYWMH 128CEA CDR-H2 FIRNKANGGTTEYAASVKG 129 CEA CDR-H3 DRGLRFYFDY 130 CEA CDR-L1TLRRGINVGAYSIY 131 CEA CDR-L2 YKSDSDKQQGSGV 132 CEA CDR-L3 MIWHSGASAV133 CEA VH EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS 134 CEA VLQAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSED EADYYCMIWHSGASAVFGGGTKLTVL135 His-tagged human FAP RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSECD ADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 136 mouse FAP UniProt accession no. P97321137 His-tagged mouse FAP RPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSECD EDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEENDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 138 His-tagged cynomolgusRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQS FAP ECDADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 139 human FolR1UniProt accession no. P15328 140 murine FolR1UniProt accession no. P35846 141 cynomolgus FolR1UniProt accession no. G7PR14 142 human MCSP UniProt accession no. Q6UVK1143 human EGFR UniProt accession no. P00533 144 human HER2Uniprot accession no. P04626 145 p95 HER2MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGT PTAENPEYLGLDVPV 146Peptide linker (G4S) GGGGS 147 Peptide linker (G4S)2 GGGGSGGGGS 148Peptide linker (SG4)2 SGGGGSGGGG 149 Peptide linker GGGGSGGGGSGGGGG4(SG4)2 150 peptide linker GSPGSSSSGS 151 (G4S)3 peptide linkerGGGGSGGGGSGGGGS 152 (G4S)4 peptide linker GGGGSGGGGSGGGGGGGGS 153peptide linker GSGSGSGS 154 peptide linker GSGSGNGS 155 peptide linkerGGSGSGSG 156 peptide linker GGSGSG 157 peptide linker GGSG 158peptide linker GGSGNGSG 159 peptide linker GGNGSGSG 160 peptide linkerGGNGSG 161 Light chain DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPK“CEA _(2F1)” LLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQ (CEA TCB)YYTYPLFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 162 Light Chain humanizedQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQA CD3 _(CH2527) (Crossfab,FRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYC VL-CH1)ALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAA (CEA TCB)LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 163 CEA _(CH1A1A 98/99)-QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGL humanized CD3 _(CH2527)EWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDD (Crossfab VH-Ck)-TAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPS Fc(knob) P329GLALASKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS (CEA TCB)SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSP 164CEA _(CH1A1A 98/99) (VH- QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLCH1)-Fc(hole) EWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDD P329GLALATAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPS (CEA TCB)SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 165 CD3 VH-CLEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGL (CEACAM5 TCB)EWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 166humanized CEA VH- QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQAPGQGLCH1(EE)-Fc (hole, P329G EWMGRIDPANGNSKYVPKFQGRVTITADTSTSTAYMELSSLRSEDLALA) TAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPS (CEACAM5 TCB)SKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 167 humanized CEA VH-QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQAPGQGL CH1(EE)-CD3 VL-CH1-EWMGRIDPANGNSKYVPKFQGRVTITADTSTSTAYMELSSLRSED Fc (knob, P329G LALA)TAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPS (CEACAM5 TCB)SKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 168 humanized CEA VL-EIVLTQSPATLSLSPGERATLSCRAGESVDIFGVGFLHWYQQKPG CL(RK)QAPRLLIYRASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY (CEACAM5 TCB)YCQQTNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 169 CEACAM5-basedQLTTESMPFNVAEGKEVLLLVHNLPQQLFGYSWYKGERVDGNRQI antigen Hu N(A2-B2)A-VGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQV avi-HisIKSDLVNEEATGQFHVYPELPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSALSPVVAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERMKLSQGNITLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENLINVDGSGLNDIFEAQ KIEWHEARAHHHHHH 170CEA (A5B7)- CDR-H1 DYYMN 171 CEA (A5B7)- CDR-H2 FIGNKANGYTTEYSASVKG 172CEA (A5B7)- CDR-H3 DRGLRFYFDY 173 CEA (A5B7)- CDR-L1 RASSSVTYIH 174CEA (A5B7)- CDR-L2 ATSNLAS 175 CEA (A5B7)- CDR-L3 QHWSSKPPT 176IgG1 Fc knob PGLALA APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 177 IgG1 Fc hole PGLALAAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSP 178 CEA (A5B7) VHEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPP (parental)GKALEWLGFIGNKANGYTTEYSASVKGRFTISRDKSQSILYLQMNTLRAEDSATYYCTRDRGLRFYFDYWGQGTTLTVSS 179 CEA (A5B7) VLQTVLSQSPAILSASPGEKVTMTCRASSSVTYIHWYQQKPGS (parental)SPKSWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDA ATYYCQHWSSKPPTFGGGTKLEIK 180CEA (A5H1EL1D)- GFTFTDYYMN CDR-H1 181 CEA (A5H1EL1D)-FIGNKANAYTTEYSASVKG CDR-H2 182 CEA (A5H1EL1D)- DRGLRFYFDY CDR-H3 183CEA (A5H1EL1D)- RASSSVTYIH CDR-L1 184 CEA (A5H1EL1D)- ATSNLAS CDR-L2 185CEA (A5H1EL1D)- QHWSSKPPT CDR-L3 186 CEA (A5H1EL1D) VHEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAP (3-23A5-1E)GKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSS 187 CEA (A5H1EL1D) VLEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQ (A5-LID)APRSWIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDF AVYYCQHWSSKPPTFGQGTKLEIK 188CEA (A5H1ELID aff. GFX₁FX₂DYX₃MN, wherein mat.) CDR-H1 X₁ is T or Y,consensus X₂ is T or S, and X₃ is Y or A or E 189 CEA (A5H1EL1D aff.X₄IX₅NKANAYTTEYSASVKG, wherein mat.) CDR-H2 X₄ is F or V, consensusX₅ is G or S 190 CEA (A5H1EL1D aff. DRGX₆RFX₇FDY, wherein mat.) CDR-H3X₆ is L or I, consensus X₇ is Y or G or Q or S 191 CEA (A5H1EL1D aff.X₈ASSSVTYIH, wherein mat.) CDR-L1 X₈ is R or H consensus 192CEA (A5H1ELID aff. ATSNLAS mat.) CDR-L2 consensus 193 CEA (A5H1EL1D aff.QHWSSX₉X₁₀PT, wherein mat.) CDR-L3 X₉ is K or V or Q or I, consensusX₁₀ is P or S 194 CEA (P006.038) VHEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYW GQGTTVTVSS 195 CEA (P006.038) VLEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSVPPTFGQGTKLEIK196 CEA (P005.097) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFSFDYW GQGTTVTVSS 197 CEA (P005.097) VLEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSQPPTFGQGTKLEIK198 CEA (P005.103) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFYFDYW GQGTTVTVSS 199 CEA (P005.103) VLEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSISPTFGQGTKLEIK200 CEA (P002.139) VH EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYAMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYW GQGTTVTVSS 201 CEA (P002.139) VLEIVLTQSPATLSLSPGERATLSCHASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSKPPTFGQGTKLEIK202 CEA (P001.177) VH EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWVRQAPGKGLEWLGFISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYW GQGTTVTVSS 203 CEA (P001.177) VLEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSKPPTFGQGTKLEIK204 CEA (P005.102) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYW GQGTTVTVSS 205 CEA (P005.102) VLEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSKSPTFGQGTKLEIK206 CEA (P005.102 EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWV combo1) VHRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYW GQGTTVTVSS 207 CEA (P005.102EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQ combo1) VLKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSKSPTFGQGTKLEIK208 CEA (P005.102 EVQLLESGGGLVQPGGSLRLSCAASGFYFSDYYMNWV combo2) VHRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYW GQGTTVTVSS 209 CEA (P005.102EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQ combo2) VLKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSKSPTFGQGTKLEIK210 CEA (P005.103 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWV combo1) VHRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFSFDYW GQGTTVTVSS 211 CEA (P005.103EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQ combo1) VLKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSISPTFGQGTKLEIK212 CEA (P005.103 EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWV combo2) VHRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFSFDYW GQGTTVTVSS 213 CEA (P005.103EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQ combo2) VLAPRSWIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDF AVYYCQHWSSISPTFGQGTKLEIK 214CEA (P006.038 EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYAMNWV combo1) VHRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYW GQGTTVTVSS 215 CEA (P006.038EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQ combo1) VLKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSVPPTFGQGTKLEIK216 CEA (P006.038 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYEMNWV combo2) VHRQAPGKGLEWLGFISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYW GQGTTVTVSS 217 CEA (P006.038EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQ combo2) VLKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQHWSSVPPTFGQGTKLEIK218 IGHV3-23-02 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAK 219 IGHV3-15*01EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKT EDTAVYYCTT 220 3-23A5-1EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKANGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS 221 3-23A5-2EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKANGYTTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS 222 3-23A5-3EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS 223 3-23A5-4EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWVGFIGNKANGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS 224 3-23A5-1AEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGL (all_backmutations)EWLGFIGNKANGYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSS 225 3-23A5-1C (A93T)EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKANGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRDRGLRFYFDYWGQGTTVTVSS 226 3-23A5-1D (K73)EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKANGYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS 227 3-15A5-1EVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKANGYTTEYSASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTRDRGLRFYFDYWGQGTTVTVSS 228 3-15A5-2EVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKANGYTTEYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTRDRGLRFYFDYWGQGTTVTVSS 229 3-15A5-3EVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGFIGNKANGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTRDRGLRFYFDYWGQGTTVTVSS 230 IGKV3-11EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ RSNWP 231 A5-L1EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW SSKPPTFGQGTKLEIK 232 A5-L2EIVLTQSPATLSLSPGERATLSCRASQSVSSYIHWYQQKPGQAPRLLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQH WSSKPPTFGQGTKLEIK 233A5-L3 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW SSKPPTFGQGTKLEIK 234 A5-L4EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQW SSKPPTFGQGTKLEIK 235A5-L1A QTVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGSSPKS (all_backmutations)WIYATSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQHW SSKPPTFGQGTKLEIK 236A5-L1B (Q1T2) QTVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW SSKPPTFGQGTKLEIK 237A5-L1C (FR2) EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGSSPKSWIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW SSKPPTFGQGTKLEIK 238NABA-avi-His See Table 15 239 N(A2B2)A-avi-His See Table 15 240NA(B2)A-avi-His See Table 15 241 A5H1EL1D_H1_rev_TN See Table 16 242A5H1EL1D_H2_for_TN See Table 16 243 LMB3 long See Table 16 244HCDR3-rev-constant See Table 16 245 A5H1EL1D_L1_rev_TN See Table 17 246A5H1EL1D_L2_for_TN See Table 17 247 A5H1EL1D_ See Table 18 L3_for_TN 248A5H1EL1D_ See Table 18 H3_rev_TN 249 LCDR3-rev-constant See Table 18 250HCDR3 amplification See Table 18 251 CEA (P006.038)- CDR- See Table 22H1 252 CEA (P006.038)- CDR- See Table 22 H2 253 CEA (P006.038)- CDR-See Table 22 H3 254 CEA (P006.038)- CDR- See Table 23 L1 255CEA (P006.038)- CDR- See Table 23 L2 256 CEA (P006.038)- CDR-See Table 23 L3 257 CEA (P005.097)- CDR- See Table 22 H1 258CEA (P005.097)- CDR- See Table 22 H2 259 CEA (P005.097)- CDR-See Table 22 H3 260 CEA (P005.097)- CDR- See Table 23 L1 261CEA (P005.097)- CDR- See Table 23 L2 262 CEA (P005.097)- CDR-See Table 23 L3 263 CEA (P005.103)- CDR- See Table 22 H1 264CEA (P005.103)- CDR- See Table 22 H2 265 CEA (P005.103)- CDR-See Table 22 H3 266 CEA (P005.103)- CDR- See Table 23 L1 267CEA (P005.103)- CDR- See Table 23 L2 268 CEA (P005.103)- CDR-See Table 23 L3 269 CEA (P002.139)- CDR- See Table 22 H1 270CEA (P002.139)- CDR- See Table 22 H2 271 CEA (P002.139)- CDR-See Table 22 H3 272 CEA (P002.139)- CDR- See Table 23 L1 273CEA (P002.139)- CDR- See Table 23 L2 274 CEA (P002.139)- CDR-See Table 23 L3 275 CEA (P001.177)- CDR- See Table 22 H1 276CEA (P001.177)- CDR- See Table 22 H2 277 CEA (P001.177)- CDR-See Table 22 H3 278 CEA (P001.177)- CDR- See Table 23 L1 279CEA (P001.177)- CDR- See Table 23 L2 280 CEA (P001.177)- CDR-See Table 23 L3 281 CEA (P005.102)- CDR- See Table 22 H1 282CEA (P005.102)- CDR- See Table 22 H2 283 CEA (P005.102)- CDR-See Table 22 H3 284 CEA (P005.102)- CDR- See Table 23 L1 285CEA (P005.102)- CDR- See Table 23 L2 286 CEA (P005.102)- CDR-See Table 23 L3 287 CEA (P005.102- See Table 22 combo1)- CDR-H1 288CEA (P005.102- See Table 22 combo1)- CDR-H2 289 CEA (P005.102-See Table 22 combo1)- CDR-H3 290 CEA (P005.102- See Table 23combo1)- CDR-L1 291 CEA (P005.102- See Table 23 combo1)- CDR-L2 292CEA (P005.102- See Table 23 combo1)- CDR-L3 293 CEA (P005.102-See Table 22 combo2)- CDR-H1 294 CEA (P005.102- See Table 22combo2)- CDR-H2 295 CEA (P005.102- See Table 22 combo2)- CDR-H3 296CEA (P005.102- See Table 23 combo2)- CDR-L1 297 CEA (P005.102-See Table 23 combo2)- CDR-L2 298 CEA (P005.102- See Table 23combo2)- CDR-L3 299 CEA (P005.103- See Table 22 combo1)- CDR-H1 300CEA (P005.103- See Table 22 combo1)- CDR-H2 301 CEA (P005.103-See Table 22 combo1)- CDR-H3 302 CEA (P005.103- See Table 23combo1)- CDR-L1 303 CEA (P005.103- See Table 23 combo1)- CDR-L2 304CEA (P005.103- See Table 23 combo1)- CDR-L3 305 CEA (P005.103-See Table 22 combo2)- CDR-H1 306 CEA (P005.103- See Table 22combo2)- CDR-H2 307 CEA (P005.103- See Table 22 combo2)- CDR-H3 308CEA (P005.103- See Table 23 combo2)- CDR-L1 309 CEA (P005.103-See Table 23 combo2)- CDR-L2 310 CEA (P005.103- See Table 23combo2)- CDR-L3 311 CEA (P006.038- See Table 22 combo1)- CDR-H1 312CEA (P006.038- See Table 22 combo1)- CDR-H2 313 CEA (P006.038-See Table 22 combo1)- CDR-H3 314 CEA (P006.038- See Table 23combo1)- CDR-L1 315 CEA (P006.038- See Table 23 combo1)- CDR-L2 316CEA (P006.038- See Table 23 combo1)- CDR-L3 317 CEA (P006.038-See Table 22 combo2)- CDR-H1 318 CEA (P006.038- See Table 22combo2)- CDR-H2 319 CEA (P006.038- See Table 22 combo2)- CDR-H3 320CEA (P006.038- See Table 23 combo2)- CDR-L1 321 CEA (P006.038-See Table 23 combo2)- CDR-L2 322 CEA (P006.038- See Table 23combo2)- CDR-L3 323 VL CEA (A5H1EL1D)- See Table 24 CH1- Fc hole PGLALA324 VH CEA (A5H1EL1D)- See Table 24 CL 325 VL CEA (P006.038) -See Table 24 CH1- Fc hole PGLALA 326 VH CEA (P006.038) - See Table 24 CL327 VL CEA (P005.097) - See Table 24 CH1- Fc hole PGLALA 328VH CEA (P005.097) - See Table 24 CL 329 VL CEA (P005.103) - See Table 24CH1- Fc hole PGLALA 330 VH CEA (P005.103) - See Table 24 CL 331VL CEA (P002.139) - See Table 24 CH1- Fc hole PGLALA 332VH CEA (P002.139) - See Table 24 CL 333 VL CEA (P001.177) - See Table 24CH1- Fc hole PGLALA 334 VH CEA (P001.177) - See Table 24 CL 335VL CEA (P005.102) - See Table 24 CH1- Fc hole PGLALA 336VH CEA (P005.102) - See Table 24 CL 337 VL CEA (P005.102 See Table 24combo1) -CH1- Fc hole PGLALA 338 VH CEA (P005.102 See Table 24combo1) - CL 339 VL CEA (P005.102 See Table 24 combo2) -CH1- Fc holePGLALA 340 VH CEA (P005.102 See Table 24 combo2) - CL 341VL CEA (P005.103 See Table 24 combo1) -CH1- Fc hole PGLALA 342VH CEA (P005.103 See Table 24 combo1) - CL 343 VL CEA (P005.103See Table 24 combo2) -CH1- Fc hole PGLALA 344 VH CEA (P005.103See Table 24 combo2) - CL 345 VL CEA (P006.038 See Table 24combo1) -CH1- Fc hole PGLALA 346 VH CEA (P006.038 See Table 24combo1) - CL 347 VL CEA (P006.038 See Table 24 combo2) -CH1- Fc holePGLALA 348 VH CEA (P006.038 See Table 24 combo2) - CL 349VH CD28 (SA Variant See Table 24 15) - CH1- Fc knob PGLALA 350VL CD28 (SA_Varaint See Table 24 15) - CL 351 CEA(A5H1EL1D) VL-EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS CH1 hu IgG1 Fc holeWIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW PGLALASSKPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 352 CEA(A5H1EL1D) VH-EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGL CkappaEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 353 CD28(SA) hu IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL VH-CH1 “EE” Fc knobEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD PGLALATAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 354 CD28(SA) hu IgG1 VL-DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK Ck “RK”LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 355 CD28(SA_Variant 8) huQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL IgG1 VH-CH1 “EE” FcEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD knob PGLALATAVYFCTRSHYGLDFNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 356 CD28(SA_Variant 8) huDIQMTQSPSSLSASVGDRVTITCHASQNIYVYLNWYQQKPGKAPK IgG1 VL-Ck “RK”LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 357 CD28(SA_Variant 15)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hu IgG1 VH-CH1 “EE”EWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD Fc knob PGLALATAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 358 CD28(SA_Variant 15)DIQMTQSPSSLSASVGDRVTITCHASQNIYVFLNWYQQKPGKAPK hu IgG1 VL-Ck “RK”LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 359 CD28(SA_Variant 29)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hu IgG1 VH-CH1 “EE”EWIGSIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD Fc knob PGLALATAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 360 CEA(A5H1EL1D) huEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGL IgG1 VH-CH1 “EE” FcEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRA hole PGLALAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 361 CEA(A5H1EL1D) huEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS IgG1 VL-Ck “RK”WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 362 CD28(SA) VL-CH1 huDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK IgG1 Fc knob PGLALALLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 363 CD28(SA) VH-CkappaQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 364 CD28(SA_Variant 8)DIQMTQSPSSLSASVGDRVTITCHASQNIYVYLNWYQQKPGKAPK VL-CH1 hu IgG1 FcLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ knob PGLALAGQTYPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 365 CD28(SA_Variant 8)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL VH-CkappaEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDFNFDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 366 CD28(SA_Variant 15)DIQMTQSPSSLSASVGDRVTITCHASQNIYVFLNWYQQKPGKAPK VL-CH1 hu IgG1 FcLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ knob PGLALAGQTYPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 367 CD28(SA_Variant 15)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL VH-CkappaEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 368 CD28(SA_Variant 29)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL VH-CkappaEWIGSIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 369 CEA(T84.66) VL-CH1EIVLTQSPATLSLSPGERATLSCRAGESVDIFGVGFLHWYQQKPG hu IgG1 Fc holeQAPRLLIYRASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY PGLALAYCQQTNEDPYTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 370 CEA(T84.66) VH-QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQAPGQGL CkappaEWMGRIDPANGNSKYVPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 371CEA(T84.66) hu IgG1 QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQAPGQGLVH-CH1 “EE” Fc hole EWMGRIDPANGNSKYVPKFQGRVTITADTSTSTAYMELSSLRSED PGLALATAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 372 CEA(T84.66) hu IgG1EIVLTQSPATLSLSPGERATLSCRAGESVDIFGVGFLHWYQQKPG VL-Ck “RK”QAPRLLIYRASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQTNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 373 CEA(A5H1EL1D) VH-EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGL CH1-VH-CH1 “EE” huEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRA IgG1 Fc hole PGLALAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP 374CD28(SA) VL-CH1 DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKCEA(A5H1EL1D) VH- LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQCH1 “EE” hu IgG1 Fc GQTYPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGknob PGLALA CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 375 CEA(P002.139) VL-EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS CH1 hu IgG1 Fc holeWIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW PGLALASSKPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 376 CEA(P002.139) VH-EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGL CkappaEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 377CD28 (SA_Variant 8) DIQMTQSPSSLSASVGDRVTITCHASQNIYVYLNWYQQKPGKAPKhu IgG1 light chain LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 378 CD28(SA_Variant 8) huQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL IgG1 PGLALA heavyEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD chainTAVYFCTRSHYGLDFNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 379 CD28 (SA_Variant 11)DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK hu IgG1 light chainLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 380 CD28(SA_Variant 11)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hu IgG1 PGLALAEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD heavy chainTAVYFCTRSHYGLDFNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 381 CD28 (SA_Variant 15)DIQMTQSPSSLSASVGDRVTITCHASQNIYVFLNWYQQKPGKAPK hu IgG1 light chainLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 382 CD28(SA_Variant 15)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hu IgG1 PGLALAEWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD heavy chainTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 383 CD28 (SA_Variant 27)DIQMTQSPSSLSASVGDRVTITCHASQGIYVYLNWYQQKPGKAPK hu IgG1 light chainLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 384 CD28(SA_Variant 27)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hu IgG1 PGLALAEWIGSIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD heavy chainTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 385 CD28 (SA_Variant 29)DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK hu IgG1 light chainLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 386 CD28(SA_Variant 29)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL hu IgG1 PGLALAEWIGSIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD heavy chainTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 387 Avi tag GLNDIFEAQKIEWHE388 CD28 VHCH1 “EE”- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL(G4S)2- FAP (4B9)- EWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDVHCH1 “EE” - Fc TAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSSknob PGLALA KSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSP 389 CD28 VLCL “RK”-DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK (G4S)2- FAP (4B9)-LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ VLCL “RK”GQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRESGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 390 EpCAM(MT201) huEVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGL IgG1 VH-CH1 “EE” FcEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAED hole PGLALATAVYYCAKDMGWGSGWRPYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSP 391 EpCAM(MT201) VL-ELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGQPPK Ckappa “RK”LLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 392 Her3 VL-CH1 hu IgG1DIVMTQSPDSLAVSLGERATINCKSSQSVLNSGNQKNYLTWYQQK Fc hole PGLALAPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQSDYSYPYTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 393 HER3 VH-CkappaQVQLVQSGAEVKKPGASVKVSCKASGYTFRSSYISWVRQAPGQGLEWMGWIYAGTGSPSYNQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARHRDYYSNSLTYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 394 CD30 VL-CH1 hu IgG1DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPG Fc hole PGLALAQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 395 CD30 VH-CkappaQIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSAASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 396 TPBG VL-CH1 huDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK IgG1 Fc hole PGLALALLIYAASTLQIGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 397 TPBG VH-CkappaEVHLLESGGGLVHPGGSLRLSCAASGFTFRSDAMHWVRQAPGKGLEWVSGVSGSGGSPYYADSVKGRFTISRDDSKTTLYLQMNSLRAEDTAVYYCATGGSIAGSYYYYPMDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 398GPRC5D (5E11) hu EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLIgG1 VH-CH1 “EE” Fc EWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDhole PGLALA TAVYYCATHTGDYFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 399 GPRC5D (5E11)VL-EIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPG Ckappa “RK”QQPKLLIYHASILASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTRESPLTFGQGTRLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 400 CD38 VL-CH1 hu IgG1EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR Fc hole PGLALALLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 401 CD38 VH-CkappaEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 402 BCMA hu IgG1 VH-EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGL CH1 “EE” Fc holeEWVSAITASGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMSLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG PGLALAGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 403 BCMA VL-CkappaEIVLTQSPGTLSLSPGERATLSCRASQSVSAYYLAWYQQKPGQAP “RK”RLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYERWPLTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 404 GPRC5D (5E11) VH-EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGL CH1(EE)-CD3 VL-CHI-EWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED Fc (knob, P329G LALA)TAVYYCATHTGDYFDYWGQGTMVTVSSASTKGPSVFPLAPSSKST (GPRC5D TCB)SGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 405 CD3 VH-CL (GPRC5DEVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGL TCB)EWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 406 CD19(8B8-2B11)DYIMH CDR-H1 407 CD19 (8B8-2B11) YINPYNDGSKYTEKFQG CDR-H2 408CD19 (8B8-2B11) GTYYYGPQLFDY CDR-H3 409 CD19(8B8-2B11) KSSQSLETSTGTTYLNCDR-L1 410 CD19 (8B8-2B11) RVSKRFS CDR-L2 411 CD19 (8B8-2B11) LQLLEDPYTCDR-L3 412 CD19 (8B8-2B11) VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSS 413 CD19 (8B8-2B11) VLDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCLQLLEDPYTFGQGTKLEIK 414CD19 (8B8-018) CDR- DYIMH H1 415 CD19 (8B8-018) CDR- YINPYNDGSKYTEKFQGH2 416 CD19 (8B8-018) CDR- GTYYYGSALFDY H3 417 CD19 (8B8-018) CDR-KSSQSLENPNGNTYLN L1 418 CD19 (8B8-018) CDR- RVSKRFS L2 419CD19 (8B8-018) CDR- LQLTHVPYT L3 420 CD19 (8B8-018) VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVTVSS 421 CD19 (8B8-018) VLDIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCLQLTHVPYTFGQGTKLEIK 422CD79b (huMA79b.v28) SYWIE CDR-H1 423 CD79b (huMA79b.v28)EILPGGGDTNYNEIFKG CDR-H2 424 CD79b (huMA79b.v28) RVPIRLDY CDR-H3 425CD79b (huMA79b.v28) KASQSVDYEGDSFLN CDR-L1 426 CD79b (huMA79b.v28)AASNLES CDR-L2 427 CD79b (huMA79b.v28) QQSNEDPLT CDR-L3 428CD79b (huMA79b.v28) EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGL VHEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTLVTVSS 429 CD79b (huMA79b.v28)DIQLTQSPSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPG VLKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSNEDPLTFGQGTKVEIK 430VL (CD19 2B11) -CH1 DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPFc hole PGLALA GQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 431 VH (CD19 2B11) CLQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 432 VL (huMA79b.v28) -DIQLTQSPSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPG CH1 Fc hole PGLALAKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 433 VH (huMA79b.v28) CLEVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 434 Human CD19UniProt accession No. P15391 435 Human CD79bUniProt accession No. P40259 436 Human CD20 UniProt accession No. P11836437 Human CD22 UniProt accession No. P20273 438 Human CD37Uniprot accession no. P11049 439 CD3-HCDR1 TYAMN 440 CD3-HCDR2RIRSKYNNYATYYADSVKG 441 CD3-HCDR3 HGNFGNSYVSWFAY 442 CD3-LCDR1GSSTGAVTTSNYAN 443 CD3-LCDR2 GTNKRAP 444 CD3-LCDR3 ALWYSNLWV 445 CD3 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 446 CD3 VLQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYC ALWYSNLWVFGGGTKLTVL 447CD20-HCDR1 YSWIN 448 CD20-HCDR2 RIFPGDGDTDYNGKFK 449 CD20-HCDR3NVFDGYWLVY 450 CD20-LCDR1 RSSKSLLHSNGITYLY 451 CD20-LCDR2 QMSNLVS 452CD20-LCDR3 AQNLELPYT 453 CD20 VHQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 454 CD20 VLDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCAQNLELPYTFGGGTKVEIK 455CD20 VH-CH1(EE)- QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLCD3 VL-CH1-Fc (knob, EWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDP329G LALA) TAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 456 CD20 VH-CH1(EE)-FcQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGL (hole, P329G LALA)EWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 457 CD20 VL-CL(RK)DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 458 CD3 VH-CLEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 459 Human PD-L1Uniprot accession no. Q9NZQ7 460 VH (PD-L1)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS 461 VL (PD-L1)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ YLYHPATFGQGTKVEIK 462VH (PD-L1) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS 463 VL (PD-L1)EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYGSLPWTFGQGTKVEIK 464human PD-1 Uniprot Q15116 465 VH (PD-1)QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS 466 VL (PD-1)EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVY YCQHSRDLPLTFGGGTKVEIK 467VH (PD-1) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAED TAVYYCATNDDYWGQGTLVTVSS468 VL (PD-1) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ SSNWPRTFGQGTKVEIK 469human EpCAM UniProt no. P16422 470 murine EpCAM UniProt no. Q99JW5 471human HER3 UniProt no. P21860 472 human CD30 UniProt no. P28908 473human TBPG UniProt no. Q13641 474 human CD38 UniProt no. P28907 475human BCMA UniProt no. Q02223 476 human GPRC5D UniProt no. Q9NZD1 477IgG CH1 domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKV 478IgG CH2 domain APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQESTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAK 479IgG CH3 domain GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG 480CH1 connector EPKSC 481 Hinge full DKTHTCPXCP with X being S or P 482Hinge middle HTCPXCP with X being S or P 483 Hinge shortCPXCP with X being S or P 484 IgG1, caucasianASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA allotypeLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 485IgG1, afroamerican ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAallotype LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 486 IgG2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 487 IgG3ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHE ALHNRFTQKSLSLSPGK 488 IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK 489CD28(variant 8) CDR- SYYIH H1 490 CD28(variant 8) CDR- SIYPGNVQTNYNEKFKDH2 49 CD28(variant 8) CDR- SHYGLDWNFDV H3 492 CD28(variant 8) CDR-HASQNIYVYLN L1 493 CD28(variant 8) CDR- KASNLHT L2 494CD28(variant 8) CDR- QQGQTYPYT L3 495 CD28(variant 15) CDR- SYYIH H1 496CD28(variant 15) CDR- SIYPGNVQTNYNEKFKD H2 497 CD28(variant 15) CDR-SHYGLDWNFDV H3 498 CD28(variant 15) CDR- HASQNIYVFLN L1 499CD28(variant 15) CDR- KASNLHT L2 500 CD28(variant 15) CDR- QQGQTYPYT L3501 CD28(variant 29) CDR- SYYIH H1 502 CD28(variant 29) CDR-SIYPGNVNTNYNEKFKD H2 503 CD28(variant 29) CDR- SHYGLDWNFDV H3 504CD28(variant 29) CDR- HASQNIYVWLN L1 505 CD28(variant 29) CDR- KASNLHTL2 506 CD28(variant 29) CDR- QQGQTYPYT L3 507 CEA (T84.66-LCHA)- DTYMHCDR-H1 508 CEA (T84.66-LCHA)- RIDPANGNSKYVPKFQG CDR-H2 509CEA (T84.66-LCHA)- FGYYVSDYAMAY CDR-H3 510 CEA (T84.66-LCHA)-RAGESVDIFGVGFLH CDR-L1 511 CEA (T84.66-LCHA)- RASNRAT CDR-L2 512CEA (T84.66-LCHA)- QQTNEDPYT CDR-L3 513 CEA (T84.66-LCHA)QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQAPGQGL VHEWMGRIDPANGNSKYVPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSS 514 CEA (T84.66-LCHA)EIVLTQSPATLSLSPGERATLSCRAGESVDIFGVGFLHWYQQKPG VLQAPRLLIYRASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQTNEDPYTFGQGTKLEIK 515EpCAM (MT201)- SYGMH CDR-H1 516 EpCAM (MT201)- VISYDGSNKYYADSVKG CDR-H2517 EpCAM (MT201)- DMGWGSGWRPYYYYGM CDR-H3 518 EpCAM (MT201)-RTSQSISSYLN CDR-L1 519 EpCAM (MT201)- WASTRES CDR-L2 520 EpCAM (MT201)-QQSYDIPYT CDR-L3 521 EpCAM (MT201) VHEVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDMGWGSGWRPYYYYGMDVWGQGTTVTVSS 522 EpCAM (MT201) VLELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDSATYYCQQ SYDIPYTFGQGTKLEIK 523HER3- CDR-H1 SSYIS 524 HER3- CDR-H2 WIYAGTGSPSYNQKLQG 525 HER3- CDR-H3HRDYYSNSL 526 HER3- CDR-L1 KSSQSVLNSGNQKNYLT 527 HER3- CDR-L2 WASTRES528 HER3- CDR-L3 QSDYSYPYT 529 HER3 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFRSSYISWVRQAPGQGLEWMGWIYAGTGSPSYNQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARHRDYYSNSLTYWGQGTLVTVSS 530 HER3 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQSDYSYPYTFGQGTKLEIK531 CD30- CDR-H1 DYYIT 532 CD30- CDR-H2 WIYPGSGNTKYNEKFKG 533CD30- CDR-H3 YGNYWF 534 CD30- CDR-L1 KASQSVDFDGDSYMN 535 CD30- CDR-L2AASNLES 536 CD30- CDR-L3 QQSNEDPWT 537 CD30 VHQIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA 538 CD30 VLDIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATY YCQQSNEDPWTFGGGTKLEIK 539TPBG(FAB091)- CDR- SDAMH H1 540 TPBG(FAB091)- CDR- GVSGSGGSPYYADSVKG H2541 TPBG(FAB091)- CDR- GGSIAGSYYYYPMDV H3 542 TPBG(FAB091)- CDR-QASQDISNYLN L1 543 TPBG(FAB091)- CDR- AASTLQI L2 544 TPBG(FAB091)- CDR-QQANSFPLT L3 545 TPBG(FAB091) VHEVHLLESGGGLVHPGGSLRLSCAASGFTFRSDAMHWVRQAPGKGLEWVSGVSGSGGSPYYADSVKGRFTISRDDSKTTLYLQMNSLRAEDTAVYYCATGGSIAGSYYYYPMDVWGQGTTVTVSS 546 TPBG(FAB091) VLDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASTLQIGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQ ANSFPLTFGGGTKVEIK 547CD38- CDR-H1 SFAMS 548 CD38- CDR-H2 AISGSGGGTYYADSVKG 549 CD38- CDR-H3DKILWFGEPVFDY 550 CD38- CDR-L1 RASQSVSSYLA 551 CD38- CDR-L2 DASNRAT 552CD38- CDR-L3 QQRSNWPPT 553 CD38 VHEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS 554 CD38 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ RSNWPPTFGQGTKVEIK 555BCMA - CDR-H1 SYAMN 556 BCMA - CDR-H2 AITASGGSTYYADSVKG 557BCMA - CDR-H3 YWPMSL 558 BCMA - CDR-L1 RASQSVSAYYLA 559 BCMA - CDR-L2DASIRAT 560 BCMA - CDR-L3 QQYERWPLT 561 BCMA VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAITASGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARYWPMSLWGQGTLVTVSS562 BCMA VL EIVLTQSPGTLSLSPGERATLSCRASQSVSAYYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYERWPLTFGQGTKVEIK 563GPRC5D (5E11) - KYAMA CDR-H1 564 GPRC5D (5E11) - SISTGGVNTYYADSVKGCDR-H2 565 GPRC5D (5E11) - HTGDYFDY CDR-H3 566 GPRC5D (5E11) -RASQSVSISGINLMN CDR-L1 567 GPRC5D (5E11) - HASILAS CDR-L2 568GPRC5D (5E11) - QQTRESPLT CDR-L3 569 GPRC5D (5E11) VH1cEVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 570 GPRC5D (5E11) VL2bEIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQTRESPLTFGQGTRLEIK 571GPRC5D (5E11) VHla EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYRDSVKARFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 572 GPRC5D (5E11) VH1bELQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYRDSVKARFTISRDNAKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 573 GPRC5D (5E11) VH1dELQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 574 GPRC5D (5E11) VLlaDIVMTQSPDSLAVSLGERATINCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQTRESPLTFGQGTRLEIK 575GPRC5D (5E11) VL1c DIVMTQSPDSLAVSLGERATINCKSSQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQTRESPLTFGQGTRLEIK 576GPRC5D (5E11) VL2a EIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPGQQPRLLIYHASILASGIPDRESGSGSGTDFTLTISRLEPEDFAVY YCQQTRESPLTFGQGTRLEIK 577GPRC5D (5E11) VL3a DIQMTQSPSSLSASVGDRVTITCRASQSVSISGINLMNWYQQKPGKQPKLLIYHASILASGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQTRESPLTFGQGTRLEIK 578GPRC5D (5E11) VL3b DIQMTQSPSSLSASVGDRVTITCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQTRESPLTFGQGTRLEIK 579GPRC5D (5F11) - NYGMA CDR-H1 580 GPRC5D (5F11) - SISTGGGNTYYRDSVKGCDR-H2 581 GPRC5D (5F11) - HDRGGLY CDR-H3 582 GPRC5D (5F11) -RSSKSLLHSNGITYVY CDR-L1 583 GPRC5D (5F11) - RMSNRAS CDR-L2 584GPRC5D (5F11) - GQLLENPYT CDR-L3 585 GPRC5D (5F11) VHlaQVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCTRHDRGGLYWGQGTMVTVSS586 GPRC5D (5F11) VH1b EVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYRDSVKGRFTISRDNAKNTLYLQMNSLRAED TAVYYCTRHDRGGLYWGQGTMVTVSS587 GPRC5D (5F11) VH1c QVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCTRHDRGGLYWGQGTMVTVSS588 GPRC5D (5F11) VHld EVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAED TAVYYCTRHDRGGLYWGQGTMVTVSS589 GPRC5D (5F11) VH2b EVQLVESGGGLVQPGGSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYRDSVKGRFTISRDNAKNTLYLQMNSLRAED TAVYYCTRHDRGGLYWGQGTMVTVSS590 GPRC5D (5F11) VH2d EVQLVESGGGLVQPGGSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAED TAVYYCTRHDRGGLYWGQGTMVTVSS591 GPRC5D (5F11) VLla DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYVYWYLQKPGQSPQVLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YHCGQLLENPYTFGQGTKLEIK 592GPRC5D (5F11) VL1b DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYVYWYLQKPGKSPQVLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YHCGQLLENPYTFGQGTKLEIK 593GPRC5D (5F11) VL2a DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYVYWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YHCGQLLENPYTFGQGTKLEIK 594GPRC5D (5F11) VL2b DIVMTQSPDSLAVSLGERATINCKSSKSLLHSNGITYVYWYQQKPGQPPKLLIYRMSNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAV YHCGQLLENPYTFGQGTKLEIK 595GPRC5D (5F11) VL2c EIVLTQSPGTLSLSPGERATLSCRASKSLLHSNGITYVYWYQQKPGQAPRLLIYRMSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAV YHCGQLLENPYTFGQGTKLEIK 596CD3 (C122) CDR-H1 SYAMN 597 CD3 (C122) CDR-H2 RIRSKYNNYATYYADSVKG 598CD3 (C122) CDR-H3 HTTFPSSYVSYYGY 599 CD3 (C122) CDR-L1 GSSTGAVTTSNYAN600 CD3 (C122) CDR-L2 GTNKRAP 601 CD3 (C122) CDR-L3 ALWYSNLWV 602CD3 (C122) VH EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSS 603 CD3 (C122) VLQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYC ALWYSNLWVFGGGTKLTVL 604CD3 (V9) CDR-H1 GYSFTGYTMN 605 CD3 (V9) CDR-H2 LINPYKGVSTYNQKFKD 606CD3 (V9) CDR-H3 SGYYGDSDWYFDV 607 CD3 (V9) CDR-L1 RASQDIRNYLN 608CD3 (V9) CDR-L2 YTSRLES 609 CD3 (V9) CDR-L3 QQGNTLPWT 610 CD3 (V9) VHEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS 611 CD3 (V9) VLDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQ GNTLPWTFGQGTKVEIK

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, MD (1991).Amino acids of antibody chains are numbered and referred to according tothe numbering systems according to Kabat (Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, ed., Public HealthService, National Institutes of Health, Bethesda, MD (1991)) as definedabove.

The following numbered paragraphs (paras) describe aspects of thepresent invention:

-   -   1. A bispecific agonistic CD28 antigen binding molecule        characterized by monovalent binding to CD28, comprising        (a) one antigen binding domain capable of specific binding to        CD28,        (b) at least one antigen binding domain capable of specific        binding to a tumor-associated antigen, and        (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.    -   2. The bispecific agonistic CD28 antigen binding molecule of        para 1, wherein the Fc domain is an IgG, particularly an IgG1 Fc        domain or an IgG4 Fc domain.    -   3. The bispecific agonistic CD28 antigen binding molecule of        paras 1 or 2, wherein the Fc domain is of human IgG1 subclass        and comprises the amino acid mutations L234A, L235A and P329G        (numbering according to Kabat EU index).    -   4. The bispecific agonistic CD28 antigen binding molecule of any        one of paras 1 to 3, wherein the antigen binding domain capable        of specific binding to CD28 comprises        (i) a heavy chain variable region (V_(H)CD28) comprising a heavy        chain complementary determining region CDR-H1 of SEQ ID NO: 20,        a CDR-H2 of SEQ ID NO: 21, and a CDR-H3 of SEQ ID NO: 22, and a        light chain variable region (V_(L)CD28) comprising a light chain        complementary determining region CDR-L1 of SEQ ID NO: 23, a        CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQ ID NO: 25; or        (ii) a heavy chain variable region (V_(H)CD28) comprising a        CDR-H1 of SEQ ID NO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3        of SEQ ID NO: 38, and a light chain variable region (V_(L)CD28)        comprising a CDR-L1 of SEQ ID NO: 39, a CDR-L2 of SEQ ID NO: 40        and a CDR-L3 of SEQ ID NO: 41.    -   5. The bispecific agonistic CD28 antigen binding molecule of any        one of paras 1 to 4, wherein the antigen binding domain capable        of specific binding to CD28 comprises a heavy chain variable        region (V_(H)CD28) comprising a CDR-H1 of SEQ ID NO: 20, a        CDR-H2 of SEQ ID NO: 21, and a CDR-H3 of SEQ ID NO: 22, and a        light chain variable region (V_(L)CD28) comprising a CDR-L1 of        SEQ ID NO: 23, a CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQ ID        NO: 25.    -   6. The bispecific agonistic CD28 antigen binding molecule of any        one of paras 1 to 5, wherein the antigen binding domain capable        of specific binding to CD28 comprises a heavy chain variable        region (V_(H)CD28) comprising an amino acid sequence that is at        least about 95%, 96%, 97%, 98%, 99% or 100% identical to the        amino acid sequence of SEQ ID NO:26, and a light chain variable        region (V_(L)CD28) comprising an amino acid sequence that is at        least about 95%, 96%, 97%, 98%, 99% or 100% identical to the        amino acid sequence of SEQ ID NO:27.    -   7. The bispecific agonistic CD28 antigen binding molecule of any        one of paras 1 to 4, wherein the antigen binding domain capable        of specific binding to CD28 comprises a heavy chain variable        region (V_(H)CD28) comprising an amino acid sequence selected        from the group consisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID        NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,        SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, and a light chain        variable region (V_(L)CD28) comprising an amino acid sequence        selected from the group consisting of SEQ ID NO:27, SEQ ID        NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,        SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and

SEQ ID NO:60.

-   -   8. The bispecific agonistic CD28 antigen binding molecule of any        one of paras 1 to 4 or 7, wherein the antigen binding domain        capable of specific binding to CD28 comprises    -   (a) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (b) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (c) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:51 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:61, or    -   (d) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (e) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (f) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (g) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:46 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (h) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:43 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (j) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (k) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27.    -   9. The bispecific agonistic CD28 antigen binding molecule of any        one of paras 1 to 8, wherein the antigen binding domain capable        of specific binding to a tumor-associated antigen is an antigen        binding domain capable of specific binding to Carcinoembryonic        Antigen (CEA).    -   10. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 9, wherein the antigen binding domain        capable of specific binding to CEA comprises a heavy chain        variable region (V_(H)CEA) comprising (i) CDR-H1 comprising the        amino acid sequence of SEQ ID NO:127, (ii) CDR-H2 comprising the        amino acid sequence of SEQ ID NO:128, and (iii) CDR-H3        comprising the amino acid sequence of SEQ ID NO:129, and a light        chain variable region (V_(L)CEA) comprising (iv) CDR-L1        comprising the amino acid sequence of SEQ ID NO:130, (v) CDR-L2        comprising the amino acid sequence of SEQ ID NO:131, and (vi)        CDR-L3 comprising the amino acid sequence of SEQ ID NO:132.    -   11. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 10, wherein the antigen binding domain        capable of specific binding to CEA comprises a heavy chain        variable region (V H CEA) comprising an amino acid sequence that        is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to        the amino acid sequence of SEQ ID NO:133, and a light chain        variable region (V_(L)CEA) comprising an amino acid sequence        that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical        to the amino acid sequence of SEQ ID NO:134.    -   12. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 8, wherein the antigen binding domain        capable of specific binding to a tumor-associated antigen is an        antigen binding domain capable of specific binding to Fibroblast        Activation Protein (FAP).    -   13. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 8 or 12, wherein the antigen binding        domain capable of specific binding to FAP comprises    -   (a) a heavy chain variable region (V_(H)FAP) comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:12, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:13,        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:14, and a light chain variable region (V_(L)FAP)        comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ        ID NO:15, (v) CDR-L2 comprising the amino acid sequence of SEQ        ID NO:16, and (vi) CDR-L3 comprising the amino acid sequence of        SEQ ID NO:17, or    -   (b) a heavy chain variable region (V_(H)FAP) comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:4, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:6, and a light chain variable region (V_(L)FAP)        comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ        ID NO:7, (v) CDR-L2 comprising the amino acid sequence of SEQ ID        NO:8, and (vi) CDR-L3 comprising the amino acid sequence of SEQ        ID NO:9.    -   14. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 8 or 12 or 13, wherein the antigen binding        domain capable of specific binding to FAP comprises        (a) a heavy chain variable region (V_(H)FAP) comprising an amino        acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or        100% identical to the amino acid sequence of SEQ ID NO:18, and a        light chain variable region (V_(L)FAP) comprising an amino acid        sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%        identical to the amino acid sequence of SEQ ID NO:19, or        (b) a heavy chain variable region (V_(H)FAP) comprising an amino        acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or        100% identical to the amino acid sequence of SEQ ID NO:10, and a        light chain variable region (V_(L)FAP) comprising an amino acid        sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%        identical to the amino acid sequence of SEQ ID NO:11.    -   15. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 8 or 12 to 14, wherein the antigen binding        domain capable of specific binding to FAP comprises a heavy        chain variable region (V_(H)FAP) comprising the amino acid        sequence of SEQ ID NO:18 and a light chain variable region        (V_(L)FAP) comprising the amino acid sequence of SEQ ID NO:19.    -   16. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 15, wherein the antigen binding domain        capable of specific binding to CD28 is a Fab fragment or a        crossFab fragment.    -   17. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 16, comprising    -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.    -   18. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 16, comprising    -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second Fab fragment capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,        wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of one of        the Fc domain subunits.    -   19. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 16, comprising    -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second and a third Fab fragment capable of specific        binding to a tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of the        first Fc domain subunit, and the third Fab fragment capable of        specific binding to a tumor-associated antigen is fused at the        C-terminus of the Fab heavy chain to the N-terminus of the        second Fc domain subunit.

The bispecific agonistic CD28 antigen binding molecule of any one ofparas 1 to 16, comprising

-   -   (a) a Fab fragment capable of specific binding to CD28,    -   (b) a VH and VL domain capable of specific binding to a        tumor-associated antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,        wherein the Fab fragment capable of specific binding to CD28 is        fused at its C-terminus to the        N-terminus of the first Fc domain subunit, and wherein one of        the VH and VL domain capable of specific binding to a        tumor-associated antigen is fused via a peptide linker to the        C-terminus of the first Fc domain subunit and the other one of        the VH and VL domain capable of specific binding to a        tumor-associated antigen is fused via a peptide linker to the        C-terminus of the second Fc domain subunit.    -   21. A polynucleotide encoding the bispecific agonistic CD28        antigen binding molecule of any one of paras 1 to 20.    -   22. A host cell comprising the polynucleotide of claim 21.    -   23. A method of producing the bispecific agonistic CD28 antigen        binding molecule of any one of paras 1 to 20 comprising        culturing the host cell of claim 22 under conditions suitable        for the expression of the bispecific antigen binding molecule.    -   24. A pharmaceutical composition comprising the bispecific        agonistic CD28 antigen binding molecule of any one of paras 1 to        20 and at least one pharmaceutically acceptable excipient.    -   25. The pharmaceutical composition of para 24 for use in the        treatment of cancer.    -   26. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 20, or the pharmaceutical composition of        para 24, for use as a medicament.    -   27. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 20, or the pharmaceutical composition of        para 24, for use in the treatment of cancer.    -   28. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 1 to 20 for use in the treatment of cancer,        wherein the agonistic CD28 antigen binding molecule is        administered in combination with a chemotherapeutic agent,        radiation therapy and/or other agents for use in cancer        immunotherapy.    -   29. Use of the bispecific agonistic CD28 antigen binding        molecule of any one of paras 1 to or the pharmaceutical        composition of para 24, in the manufacture of a medicament for        the treatment of cancer.    -   30. A method of inhibiting the growth of tumor cells in an        individual comprising administering to the individual an        effective amount of the bispecific agonistic CD28 antigen        binding molecule of any one of paras 1 to 20, or the        pharmaceutical composition of para 24, to inhibit the growth of        the tumor cells.    -   31. A method of treating cancer comprising administering to the        individual a therapeutically effective amount of the bispecific        agonistic CD28 antigen binding molecule of any one of paras 1 to        20, or the pharmaceutical composition of para 24.    -   32. A bispecific agonistic CD28 antigen binding molecule        comprising an antigen binding domain capable of specific binding        to CD28, an antigen binding domain capable of specific binding        to a B cell surface antigen, and a Fc domain composed of a first        and a second subunit capable of stable association comprising        one or more amino acid substitution that reduces the binding        affinity of the antigen binding molecule to an Fc receptor        and/or effector function.    -   33. The bispecific agonistic CD28 antigen binding molecule of        para 32, characterized by monovalent binding to CD28.    -   34. The bispecific agonistic CD28 antigen binding molecule of        para 32, further characterized by monovalent binding to the B        cell surface antigen.    -   35. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 34, wherein the Fc domain is of human        IgG1 subclass and comprises the amino acid mutations L234A,        L235A and P329G (numbering according to Kabat EU index).    -   36. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 35, wherein the antigen binding domain        capable of specific binding to CD28 comprises        (i) a heavy chain variable region (V_(H)CD28) comprising a heavy        chain complementary determining region CDR-H1 of SEQ ID NO: 20,        a CDR-H2 of SEQ ID NO: 21, and a CDR-H3 of SEQ ID NO: 22, and a        light chain variable region (V_(L)CD28) comprising a light chain        complementary determining region CDR-L1 of SEQ ID NO: 23, a        CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQ ID NO: 25; or        (ii) a heavy chain variable region (V_(H)CD28) comprising a        CDR-H1 of SEQ ID NO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3        of SEQ ID NO: 38, and a light chain variable region (V_(L)CD28)        comprising a CDR-L1 of SEQ ID NO: 39, a CDR-L2 of SEQ ID NO: 40        and a CDR-L3 of SEQ ID NO: 41.    -   37. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 36, wherein the antigen binding domain        capable of specific binding to CD28 comprises a heavy chain        variable region (V_(H)CD28) comprising a CDR-H1 of SEQ ID NO:20,        a CDR-H2 of SEQ ID NO:21, and a CDR-H3 of SEQ ID NO:22, and a        light chain variable region (V_(L)CD28) comprising a CDR-L1 of        SEQ ID NO:23, a CDR-L2 of SEQ ID NO: 24 and a CDR-L3 of SEQ ID        NO:25.    -   38. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 37, wherein the antigen binding domain        capable of specific binding to CD28 comprises a heavy chain        variable region (V_(H)CD28) comprising an amino acid sequence        that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical        to the amino acid sequence of SEQ ID NO:26, and a light chain        variable region (V_(L)CD28) comprising an amino acid sequence        that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical        to the amino acid sequence of SEQ ID NO:27.    -   39. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 36, wherein the antigen binding domain        capable of specific binding to CD28 comprises a heavy chain        variable region (V_(H)CD28) comprising an amino acid sequence        selected from the group consisting of SEQ ID NO:42, SEQ ID        NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,        SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, and a        light chain variable region (V_(L)CD28) comprising an amino acid        sequence selected from the group consisting of SEQ ID NO:27, SEQ        ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID        NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60        and SEQ ID NO:61.    -   40. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 36 or 39, wherein the antigen binding        domain capable of specific binding to CD28 comprises    -   (a) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (b) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (c) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:51 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:61, or    -   (d) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:48 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (e) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:48 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or    -   (f) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:48 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (g) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:48 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (h) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:43 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27, or    -   (i) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:53, or    -   (j) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:59, or    -   (k) a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:42 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:27.    -   41. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 36 or 39 or 40, wherein the antigen        binding domain capable of specific binding to CD28 comprises a        heavy chain variable region (V_(H)CD28) comprising the amino        acid sequence of SEQ ID NO:46 and a light chain variable region        (V_(L)CD28) comprising the amino acid sequence of SEQ ID NO:53,        or a heavy chain variable region (V_(H)CD28) comprising the        amino acid sequence of SEQ ID NO:47 and a light chain variable        region (V_(L)CD28) comprising the amino acid sequence of SEQ ID        NO:54, or a heavy chain variable region (V_(H)CD28) comprising        the amino acid sequence of SEQ ID NO:47 and a light chain        variable region (V_(L)CD28) comprising the amino acid sequence        of SEQ ID NO:9, or a heavy chain variable region (V_(H)CD28)        comprising the amino acid sequence of SEQ ID NO:16 and a light        chain variable region (V_(L)CD28) comprising the amino acid        sequence of SEQ ID NO:9.    -   42. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 41, wherein the B cell surface antigen is        selected from the group consisting of CD19, CD79b, CD20, CD22        and CD37.    -   43. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 42, wherein the antigen binding domain        capable of specific binding to a B cell surface antigen is an        antigen binding domain capable of specific binding to CD19.    -   44. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 43, wherein the antigen binding domain        capable of specific binding to CD19 comprises        (a) a heavy chain variable region (V_(H)CD19) comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:406, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:407,        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:408, and a light chain variable region (V_(L)CD19)        comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ        ID NO:409, (v) CDR-L2 comprising the amino acid sequence of SEQ        ID NO:410, and (vi) CDR-L3 comprising the amino acid sequence of        SEQ ID NO:411, or        (b) a heavy chain variable region (V_(H)CD19) comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:414, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:415,        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:416, and a light chain variable region (V_(L)CD19)        comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ        ID NO:417, (v) CDR-L2 comprising the amino acid sequence of SEQ        ID NO:418, and (vi) CDR-L3 comprising the amino acid sequence of        SEQ ID NO:419.    -   45. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 44, wherein the antigen binding domain        capable of specific binding to CD19 comprises        (a) a heavy chain variable region (V_(H)CD19) comprising an        amino acid sequence that is at least about 95%, 98% or 100%        identical to the amino acid sequence of SEQ ID NO:412, and a        light chain variable region (V_(L)CD19) comprising an amino acid        sequence that is at least about 95%, 98% or 100% identical to        the amino acid sequence of SEQ ID NO:413, or        (b) a heavy chain variable region (V_(H)CD19) comprising an        amino acid sequence that is at least about 95%, 98% or 100%        identical to the amino acid sequence of SEQ ID NO:420, and a        light chain variable region (V_(L)CD19) comprising an amino acid        sequence that is at least about 95%, 98% or 100% identical to        the amino acid sequence of SEQ ID NO:421.    -   46. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 45, wherein the antigen binding domain        capable of specific binding to CD19 comprises a heavy chain        variable region (V_(H)CD19) comprising an amino acid sequence of        SEQ ID NO:412 and a light chain variable region (V_(L)CD19)        comprising an amino acid sequence of SEQ ID NO:413.    -   47. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 43, wherein the antigen binding domain        capable of specific binding to a B cell surface antigen is an        antigen binding domain capable of specific binding to CD79b.    -   48. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 43 or 47, wherein the antigen binding        domain capable of specific binding to CD79b comprises a heavy        chain variable region (V_(H)CD79b) comprising (i) CDR-H1        comprising the amino acid sequence of SEQ ID NO:422, (ii) CDR-H2        comprising the amino acid sequence of SEQ ID NO:423, and (iii)        CDR-H3 comprising the amino acid sequence of SEQ ID NO:424, and        a light chain variable region (V_(L)CD79b) comprising (iv)        CDR-L1 comprising the amino acid sequence of SEQ ID NO:425, (v)        CDR-L2 comprising the amino acid sequence of SEQ ID NO:426,        and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID        NO:427.    -   49. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 43 or 47 or 48, wherein the antigen        binding domain capable of specific binding to CD79b comprises a        heavy chain variable region (V_(H)CD79b) comprising an amino        acid sequence that is at least about 95%, 98%, or 100% identical        to the amino acid sequence of SEQ ID NO:428, and a light chain        variable region (V_(L)CD79b) comprising an amino acid sequence        that is at least about 95%, 98% or 100% identical to the amino        acid sequence of SEQ ID NO:429.    -   50. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 49, comprising    -   (a) one Fab fragment capable of specific binding to CD28,    -   (b) one crossFab fragment capable of specific binding to a B        cell surface antigen, and    -   (c) Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function.    -   51. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 49, comprising    -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second Fab fragment capable of specific binding to a B        cell surface antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of one of        the Fc domain subunits.    -   52. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 49, comprising    -   (a) a first Fab fragment capable of specific binding to CD28,    -   (b) a second and a third Fab fragment capable of specific        binding to a B cell surface antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association comprising one or more amino acid        substitution that reduces the binding affinity of the antigen        binding molecule to an Fc receptor and/or effector function,    -   wherein the first Fab fragment capable of specific binding to        CD28 is fused at the C-terminus of the Fab heavy chain to the        N-terminus of the Fab heavy chain of the second Fab fragment        capable of specific binding to a tumor-associated antigen, which        is in turn fused at its C-terminus to the N-terminus of the        first Fc domain subunit, and the third Fab fragment capable of        specific binding to a tumor-associated antigen is fused at the        C-terminus of the Fab heavy chain to the N-terminus of the        second Fc domain subunit.    -   53. A pharmaceutical composition comprising the bispecific        agonistic CD28 antigen binding molecule of any one of paras 32        to 52 and at least one pharmaceutically acceptable excipient.    -   54. A polynucleotide encoding the bispecific agonistic CD28        antigen binding molecule of any one of paras 32 to 52.    -   55. A vector comprising the polynucleotide of para 54.    -   56. A host cell comprising the vector of para 55 or the        polynucleotide of para 54.    -   57. A method of producing the bispecific agonistic CD28 antigen        binding molecule of any one of paras 32 to 52 comprising        culturing the host cell of para 25 under conditions suitable for        the expression of the bispecific antigen binding molecule.    -   58. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 52, or the pharmaceutical composition of        para 53, for use as medicament.    -   59. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 52, or the pharmaceutical composition of        para 53, for use in the treatment of cancer.    -   60. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 52 for use in the treatment of cancer,        wherein the agonistic CD28 antigen binding molecule is for use        in combination with a chemotherapeutic agent, radiation therapy        and/or other agents for use in cancer immunotherapy.    -   60. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 52 for use in the treatment of cancer,        wherein the agonistic CD28 antigen binding molecule is for use        in combination with a T-cell activating anti-CD3 bispecific        antibody.    -   61. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 52 for use in the treatment of cancer,        wherein the agonistic CD28 antigen binding molecule is for use        in combination with an anti-CD20/anti-CD3 bispecific antibody.    -   62. The bispecific agonistic CD28 antigen binding molecule of        any one of paras 32 to 52 for use in the treatment of cancer,        wherein the agonistic CD28 antigen binding molecule is for use        in combination with an agent blocking PD-L1/PD-1 interaction.    -   63. Use of the bispecific agonistic CD28 antigen binding        molecule of any one of paras 32 to 52, or the pharmaceutical        composition of para 53, in the manufacture of a medicament for        the treatment of cancer.    -   64. A method of inhibiting the growth of tumor cells in an        individual comprising administering to the individual an        effective amount of the bispecific agonistic CD28 antigen        binding molecule of any one of paras 32 to 52, or the        pharmaceutical composition of para 53, to inhibit the growth of        the tumor cells.    -   65. A method of treating cancer comprising administering to the        individual a therapeutically effective amount of the bispecific        agonistic CD28 antigen binding molecule of any one of paras 32        to 52, or the pharmaceutical composition of para 53.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulin light and heavy chains is given in: Kabat, E. A. etal., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed.,NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments, where required, were either generated by PCRusing appropriate templates or were synthesized at Geneart AG(Regensburg, Germany) or Genscript (New Jersey, USA) from syntheticoligonucleotides and PCR products by automated gene synthesis. The genesegments flanked by singular restriction endonuclease cleavage siteswere cloned into standard cloning/sequencing vectors. The plasmid DNAwas purified from transformed bacteria and concentration determined byUV spectroscopy. The DNA sequence of the subcloned gene fragments wasconfirmed by DNA sequencing. Gene segments were designed with suitablerestriction sites to allow subcloning into the respective expressionvectors. All constructs were designed with a 5′-end DNA sequence codingfor a leader peptide which targets proteins for secretion in eukaryoticcells.

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2× PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecificantibodies with VH/VL exchange (VH/VL CrossMabs) with emphasis on theircorrect assembly. The expected primary structures were analyzed byelectrospray ionization mass spectrometry (ESI-MS) of the deglycosylatedintact CrossMabs and deglycosylated/plasmin digested or alternativelydeglycosylated/limited LysC digested CrossMabs.

The VH/VL CrossMabs were deglycosylated with N-Glycosidase F in aphosphate or Tris buffer at 37° C. for up to 17 h at a proteinconcentration of 1 mg/ml. The plasmin or limited LysC (Roche) digestionswere performed with 100 μg is deglycosylated VH/VL CrossMabs in a Trisbuffer pH 8 at room temperature for 120 hours and at 37° C. for 40 min,respectively. Prior to mass spectrometry the samples were desalted viaHPLC on a Sephadex G25 column (GE Healthcare). The total mass wasdetermined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)equipped with a TriVersa NanoMate source (Advion).

Determination of Binding and Binding Affinity of MultispecificAntibodies to the Respective Antigens Using Surface Plasmon Resonance(SPR) (BIACORE)

Binding of the generated antibodies to the respective antigens isinvestigated by surface plasmon resonance using a BIACORE instrument (GEHealthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinitymeasurements Goat-Anti-Human IgG, JIR 109-005-098 antibodies areimmobilized on a CM5 chip via amine coupling for presentation of theantibodies against the respective antigen. Binding is measured in HBSbuffer (HBS-P (10 mM HEPES, 150 mM NaCl, Tween 20, ph 7.4), 25° C. (oralternatively at 37° C.). Antigen (R&D Systems or in house purified) wasadded in various concentrations in solution. Association was measured byan antigen injection of 80 seconds to 3 minutes; dissociation wasmeasured by washing the chip surface with HBS buffer for 3-10 minutesand a KD value was estimated using a 1:1 Langmuir binding model.Negative control data (e.g. buffer curves) are subtracted from samplecurves for correction of system intrinsic baseline drift and for noisesignal reduction. The respective Biacore Evaluation Software is used foranalysis of sensorgrams and for calculation of affinity data.

Example 1

Generation and Production of Bispecific Antigen Binding MoleculesTargeting CD28 and Fibroblast Activation Protein (FAP) orCarcinoembryonic Antigen (CEA)

1.1 Cloning of Bispecific Antigen Binding Molecules Targeting CD28 andFibroblast Activation Protein (FAP) or Carcinoembryonic Antigen (CEA)

Cloning of the Antigen:

A DNA fragment encoding the extracellular domain (amino acids 1 to 134of matured protein) of human CD28 (Uniprot: P10747) was inserted inframe into two different mammalian recipient vectors upstream of afragment encoding a hum IgG1 Fc fragment which serves as solubility- andpurification tag. One of the expression vectors contained the “hole”mutations in the Fc region, the other one the “knob” mutations as wellas a C-terminal avi tag (GLNDIFEAQKIEWHE, SEQ ID NO:387) allowingspecific biotinylation during co-expression with Bir A biotin ligase. Inaddition, both Fc fragments contained the PG-LALA mutations. Bothvectors were co-transfected in combination with a plasmid coding for theBirA biotin ligase in order to get a dimeric CD28-Fc construct with amonovalent biotinylated avi-tag at the C-terminal end of the Fc-knobchain.

The variable domains of the FAP clone 4B9, a CEA binder and the CD28clones SA and mAb 9.3 were used for the generation of various tumortargeted CD28 constructs. The generation and preparation of FAP clone4B9 is disclosed in WO 2012/020006 A2, which is incorporated herein byreference. The CEA clone used in the molecules is described in WO2007/071422 and the CD28 superagonistic antibody (SA) with a VHcomprising the amino acid sequence of SEQ ID NO:26 and a VL comprisingthe amino acid sequence of SEQ ID NO:27 is described in WO 2006/050949.A description of antibody mAb 9.3 can be found in Tan et al. J.Immunology 2002, 169, 1119-1125. For the generation of the respectiveexpression plasmids, the sequences of the respective variable domainswere used and sub-cloned in frame with the respective constant regionswhich are pre-inserted in the respective recipient mammalian expressionvector. A schematic description of the resulting molecules is shown inFIGS. 1A to 1M. Where indicated, Pro329Gly, Leu234Ala and Leu235Alamutations (PG-LALA) have been introduced in the constant region of thehuman IgG1 heavy chains to abrogate binding to Fc gamma receptors. Forthe generation of unsymmetric bispecific antibodies, Fc-fragmentscontained either the “knob” or “hole” mutations to avoid mispairing ofthe heavy chains. In order to avoid mispairing of light chains in bi-and multispecific antibody constructs, exchange of VH/VL or CH1/Ckappadomains was introduced in one binding moiety (CrossFab technology). Inanother binding moiety, charges were introduced into the CH1 and Ckappadomains.

The following molecules were cloned, a schematic illustration thereof isshown in FIGS. 1A to 1M:

-   -   Molecule A: CD28(SA) (hu IgG4), TGN1412, CD28 (SA) antibody in a        human IgG4 isotype (FIG. 1A), comprises the amino acid sequences        of SEQ ID NO:62 and SEQ ID NO:63 (P1AE1975).    -   Molecule B: CD28(SA) (PG-LALA), CD28 (SA) antibody in a huIgG1        PG-LALA isotype (FIG. 1B) comprises the amino acid sequences of        SEQ ID NO:62 and SEQ ID NO:64 (P1AD9289).    -   Molecule C: FAP(4B9)-CD28(SA) 1+1 format, bispecific huIgG1        PG-LALA CrossFab molecule with charged modifications in the        CD28(SA) Fab fragment (knob) and VH/VL exchange in FAP(4B9) Fab        fragment (hole) (FIG. 1C) comprising the amino acid sequences of        SEQ ID NOs: 65, 66, 67 and 68 (P1AD4492).    -   Molecule D: FAP(4B9)-CD28(SA) 1+4 format, bispecific tetravalent        anti-CD28 (SA) and monovalent anti-FAP huIgG1 PG-LALA construct.        The VH and VL domains of the FAP clone 4B9 were fused to the        C-terminal end of respective chains of the Fc domain (VH: knob        chain, VL: hole chain) (FIG. 1F). The molecule comprises the        amino acid sequences of SEQ ID NOs: 62, 69 and 70 (P1AD9018).    -   Molecule E: FAP(4B9)-CD28(SA) 1+2 format, bispecific bivalent        anti-CD28 (SA) and monovalent anti-FAP huIgG1 PG-LALA construct.        The VH and VL domains of the FAP clone 4B9 were fused to the        C-terminal end of respective chains of the Fc domain (VH: knob        chain, VL: hole chain) (FIG. 1D). The molecule comprises the        amino acid sequences of SEQ ID NOs: 62, 71 and 72 (P1AD9011).    -   Molecule F: FAP(4B9)-CD28(SA) 2+2, bispecific bivalent anti-CD28        (SA) and bivalent anti-FAP huIgG1 PG-LALA CrossFab construct,        charged modifications in the anti-CD28 Fab fragments, VH fusion        of the anti-FAP CrossFab fragments with CH1/Ckappa exchange to        the C-terminal end of the Fc fragment (FIG. 1E). The molecule        comprises the amino acid sequences of SEQ ID NOs:65, 73 and 74        (P1AD4493).    -   Molecule G: FAP (4B9)-CD28 (SA) 2+1, bispecific monovalent        anti-CD28 (SA) and bivalent anti-FAP huIgG1 PG-LALA CrossFab        construct, “classical orientation”, VH/VL exchange in the        anti-CD28 CrossFab fragment, charged modification in anti-FAP        Fab fragments (FIG. 1L). The molecule comprises the amino acid        sequences of SEQ ID NOs: 75, 76, 77 and 78 (P1AD5231).    -   Molecule H: FAP(4B9)-CD28(SA)C-01, 1+1 bispecific monovalent        anti-CD28 (SA) and monovalent anti-FAP huIgG1 PG-LALA CrossFab        molecule, “head-to-tail”, VH/VL exchange in anti-CD28 CrossFab        fragment, charged modification in anti-FAP binder (FIG. 1M). The        molecule comprises the amino acid sequences of SEQ ID NOs: 75,        77, 78 and 79 (P1AE2021).    -   Molecule I: FAP(4B9)-CD28(SA)C-04, 1+1 bispecific monovalent        anti-CD28 (SA) and monovalent anti-FAP huIgG1 PG-LALA construct.        The VH and VL domains of the FAP binder 4B9 were fused to the        C-terminal end of respective chains of the Fc fragment (VH: knob        chain, VL: hole chain) (FIG. 1K). The molecule comprises the        amino acid sequences of SEQ ID NOs: SEQ ID NO: 62, 72 and 80        (P1AE2236).    -   Molecule J: CEA-CD28(SA) 2+2, bispecific bivalent anti-CD28 (SA)        and bivalent anti-CEA huIgG1 PG-LALA CrossFab construct, charged        modifications in the anti-CD28 Fab fragments, VH fusion of the        anti-CEA CrossFab fragment with CH1/Ckappa exchange to the        C-terminal end of the Fc fragment (FIG. 1H). The molecule        comprises the amino acid sequences of SEQ ID NOs: 65, 81 and 82        (P1AE1195).    -   Molecule K: CEA-CD28(SA) 1+2, bispecific bivalent anti-CD28 (SA)        and monovalent anti-CEA huIgG1 PG-LALA construct. The VH and VL        domains of the CEA binder were fused to the C-terminal end of        respective chains of the Fc fragment (VH: knob chain, VL: hole        chain) (FIG. 1G). The molecule comprises the amino acid        sequences of SEQ ID NOs: 62, 83 and 84 (P1AE1194).    -   Molecule L: monovalent IgG CD28 (SA), monovalent anti-CD28 (SA)        huIgG1 PG-LALA construct, wherein the CD28 heavy chain is        expressed as a “hole” Fc chain in combination with a Fc (knob)        fragment (FIG. 1I). The molecule comprises the amino acid        sequences of SEQ ID NOs: 65, 85 and 86 (P1AD8944).    -   Molecule M: CEA-CD28(SA) 1+1 format, bispecific huIgG1 PG-LALA        CrossFab molecule with charged modifications in the CD28(SA) Fab        fragment (knob) and VH/VL exchange in CEA crossFab fragment        (hole) (FIG. 1J) comprising the amino acid sequences of SEQ ID        NOs: 65, 66, 87 and 88 (P1AE3127).    -   Molecule N: mab 9.3 (PG-LALA), mAb9.3 clone in human IgG1        PG-LALA isotype (as in FIG. 1B). The molecule comprises the        amino acid sequences of SEQ ID NOs: 89 and 90 (P1 AD5142).    -   Molecule O: FAP(4B9)-CD28(mAb9.3)C-03, bispecific huIgG1 PG-LALA        CrossFab construct with charged modifications in the mAb9.3 Fab        fragment (knob) and VH/VL exchange in the anti-FAP fragment        (hole) (as in FIG. 1C). The molecule comprises the amino acid        sequences of SEQ ID NOs: 67, 68, 91 and 92 (P1AE2238).    -   Molecule P: FAP(4B9)-CD28(mAb9.3) 1+4, bispecific tetravalent        anti-CD28 mAb9.3 and anti-FAP huIgG1 PG-LALA construct. The VH        and VL domains of the FAP binder are fused to the C-terminal end        of respective chains of the Fc fragment (VH: knob chain, VL:        hole chain) (as in FIG. 1F). The molecule comprises the amino        acid sequences of SEQ ID NOs: 89, 93 and 94 (P1AD8969).    -   Molecule Q: FAP(4B9)-CD28(mAb9.3) 1+2, bispecific bivalent        anti-CD28 mAb9.3 and monovalent anti-FAP huIgG1 PG-LALA        construct. The VH and VL domains of the FAP binder were fused to        the C-terminal end of respective chains of the Fc fragment (VH:        knob chain, VL: hole chain) (as in FIG. 1D). The molecule        comprises the amino acid sequences of SEQ ID Nos: 89, 95 and 96        (P1AD8962).    -   Molecule R: FAP(4B9)-CD28(mAb9.3) 2+2, bispecific bivalent        anti-CD28 mAb9.3 and bivalent anti-FAP huIgG1 PG-LALA CrossFab        construct, charged modifications in the mAb9.3 FAP fragment, VH        fusion of the anti-FAP Fab fragment with CH1/Ckappa CrossFab        exchange to the C-terminal end of the Fc fragment (as in FIG.        1E). The molecule comprises the amino acid sequences of SEQ ID        Nos: 97, 98 and 99 (P1AD8968).    -   Molecule S: FAP (4B9)-CD28(mAb9.3) 2+1, bispecific monovalent        anti-CD28 (mAb9.3) and bivalent anti-FAP huIgG1 PG-LALA CrossFab        construct, “classical orientation”, VH/VL exchange in the        anti-CD28 (mAb9.3) CrossFab fragment, charged modification in        anti-FAP Fab fragments (as in FIG. 1L). The molecule comprises        the amino acid sequences of SEQ ID Nos: 76, 77, 100 and 101        (P1AD5560).    -   Molecule T: FAP(4B9)-CD28(mAb9.3)C-02, bispecific monovalent        anti-CD28 (mAb9.3) and monovalent anti-FAP huIgG1 PG-LALA        CrossFab construct, “head-to-tail”, VH/VL exchange in the        anti-CD28 (mAb9.3) CrossFab fragment, charged modification in        the anti-FAP fragment (as in FIG. 1M). The molecule comprises        the amino acid sequences of SEQ ID Nos: 78, 79, 100 and 101        (P1AE2022).    -   Molecule U: FAP(4B9)-CD28(mAb9.3)C-05, bispecific monovalent        anti-CD28 (mAb9.3) and monovalent anti-FAP huIgG1 PG-LALA        construct. The VH and VL domains of the FAP binder 4B9 were        fused to the C-terminal end of respective chains of the Fc        fragment (VH: Fc knob chain, VL: Fc hole chain) (as in FIG. 1K).        The molecule comprises the amino acid sequences of SEQ ID Nos:        80, 89 and 96 (P1AE2237).    -   Molecule V: CEA-CD28(mAb9.3) 2+2, bispecific bivalent anti-CD28        (mAb9.3) and bivalent anti-CEA huIgG1 PG-LALA CrossFab        construct, charged modifications in the mAb9.3 Fab fragment, VH        fusion of the anti-CEA CrossFab fragment with CH1/Ckappa        exchange to the C-terminal end of the Fc fragment (as in FIG.        1H). The molecule comprises the amino acid sequences of SEQ ID        Nos: 82, 89 and 102 (P1AE1193).    -   Molecule W: CEA-CD28(mAb9.3) 1+2, bispecific bivalent anti-CD28        (mAb9.3) and monovalent anti-CEA huIgG1 PG-LALA construct. The        VH and VL domains of the CEA binder were fused to the C-terminal        end of respective chains of the Fc fragment (VH: knob chain, VL:        hole chain) (as in FIG. 1G). The molecule comprises the amino        acid sequences of SEQ ID Nos: 89, 103 and 104 (P1AE1192).    -   Molecule X: monovalent IgG CD28 (mAb9.3), wherein the CD28 heavy        chain is expressed as a “hole” Fc chain in combination with a Fc        (knob) fragment (as in FIG. 1I). The molecule comprises the        amino acid sequences of SEQ ID Nos: 86, 105 and 106 (P1AD8938).    -   Furthermore, a trispecific molecule was prepared:    -   Molecule Y, FAP (4B9)-CD28(TGN1412)-CEA 1+1+1, trispecific        monovalent anti-CD28 (TGN1412), monovalent anti-FAP and        monovalent anti-CEA huIgG1 PG-LALA CrossFab construct, VH/VL        exchange in the anti-CEA CrossFab fragment (hole), charged        modifications in the anti-FAP Fab fragment (knob) and in the        anti-CD28 fragment (knob) (as in FIG. 1N). The molecule        comprises the amino acid sequences of SEQ ID Nos: 87, 88, 388        and 389 (P1AE4064).

1.2 Production of Bispecific Antigen Binding Molecules Targeting CD28and Fibroblast Activation Protein (FAP) or Carcinoembryonic Antigen(CEA)

Expression of the above-mentioned molecules is either driven by achimeric MPSV promoter or a CMV promoter. Polyadenylation is driven by asynthetic polyA signal sequence located at the 3′ end of the CDS. Inaddition, each vector contains an EBV OriP sequence for autosomalreplication.

For the production of the constructs C to W, HEK293-EBNA cells that growin suspension were co-transfected with the respective expression vectorsusing polyethylenimine as a transfection reagent. Antibodies andbispecific antibodies were generated by transient transfection of HEK293EBNA cells. Cells were centrifuged and medium replaced by pre-warmed CDCHO medium. Expression vectors were mixed in CD CHO medium, PEI wasadded, the solution vortexed and incubated for 10 minutes at roomtemperature. Afterwards, cells were mixed with the DNA/PEI solution,transferred to shake flask and incubated for 3 hours at 37° C. in anincubator with a 5% CO₂ atmosphere. After the incubation, Excell mediumwith supplements was added (Mammalian Cell Cultures for BiologicsManufacturing, Editors: Weichang Zhou, Anne Kantardjieff). One day aftertransfection supplements (Feed) were added (Mammalian Cell Cultures forBiologics Manufacturing, Editors: Weichang Zhou, Anne Kantardjieff).Cell supernatants were harvested after 7 days by centrifugation andsubsequent filtration (0.2 μm filter) and purified by standard methods.

Constructs A, B and X were prepared by Evitria using their proprietaryvector system with conventional (non-PCR based) cloning techniques andusing suspension-adapted CHO K1 cells (originally received from ATCC andadapted to serum-free growth in suspension culture at Evitria). For theproduction, Evitria used its proprietary, animal-component free andserum-free media (eviGrow and eviMake2) and its proprietary transfectionreagent (eviFect). Supernatant was harvested by centrifugation andsubsequent filtration (0.2 μm filter) and purified by standard methods.

1.3 Purification of Bispecific Antigen Binding Molecules Targeting CD28and Fibroblast Activation Protein (FAP) or Carcinoembryonic Antigen(CEA)

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, Fc-containing proteins were purifiedfrom cell culture supernatants by affinity chromatography using ProteinA. Elution was achieved at pH 3.0 followed by immediate neutralizationof the sample. The protein was concentrated and aggregated protein wasseparated from monomeric protein by size exclusion chromatography in 20mM histidine, 140 mM sodium chloride, pH 6.0.

1.4 Analytical Data of Bispecific or Trispecific Antibodies TargetingCD28 and Fibroblast Activation Protein (FAP) or Carcinoembryonic Antigen(CEA)

The protein concentration of purified constructs was determined bymeasuring the optical density (OD) at 280 nm, using the mass extinctioncoefficient calculated on the basis of the amino acid sequence accordingto Pace, et al., Protein Science, 1995, 4, 2411-1423. Purity andmolecular weight of the proteins were analyzed by CE-SDS in the presenceand absence of a reducing agent using a LabChipGXII (Perkin Elmer).Determination of the aggregate content was performed by HPLCchromatography at 25° C. using analytical size-exclusion column (TSKgelG3000 SW XL or UP-SW3000) equilibrated in running buffer (25 mM K₂HPO₄,125 mM NaCl, 200 mM L-Arginine Monohydrocloride, pH 6.7 or 200 mMKH₂PO₄, 250 mM KCl pH 6.2 respectively). A summary of the purificationparameters of all molecules is given in Table 1.

TABLE 1 Summary of the production and purification of bispecific ortrispecific CD28 antigen binding molecules Analytical SEC Yield(HMW/Monomer/LMW) Purity measured Molecule Description [mg/l] [%] byCE-SDS [%] A CD28(SA) 257 0/100/0 84.25 (hu IgG4) B CD28(SA) 3900/97.3/2.7 84 hu IgG1 (PG-LALA) C FAP(4B9)-CD28(SA) 19.5 0.64/97.28/2.0798.75 1 + 1 D FAP(4B9)- 1.75 3.53/96.48/0 n.d. CD28(TGN1412) 1 + 4 EFAP(4B9)-CD28(SA) 0.38 0.8/95.48/3.72 93.58 1 + 2 F FAP(4B9)-CD28(SA)18.2 1.4/98.6/0 91.42 2+2 G FAP (4B9)-CD28 (SA) 2.66 3.79/94. 02/2.19 642+1 H FAP(4B9) - CD28(SA) 10.6 0/100/0 99.38 C-01 I FAP(4B9) - CD28(SA)5.55 4.12/81.17/14.71 96.5 C-04 J CEA-CD28(SA) 2 + 2 6.25 1/99/0 n.d. KCEA-CD28(SA) 1 + 2 5.8 0.5/99.5/0 64 L monovalent IgG1 CD28 38.50.2/99.6/0.2 99.3 (SA) M CEA-CD28(SA) 1 + 1 14.3 0/100/0 99.18 N CD28(mAb 9.3) 22.06 0 /100/0 88 hu IgG1 (PG-LALA) O FAP(4B9) - 2.14 0/100/097.4 CD28(mAb9.3) C-03 P FAP(4B9)- 7.6 1.2/98.8/0 97.6 CD28(mAb9.3) 1 +4 Q FAP(4B9)- 16 1/98.5/0.5 97.16 CD28(mAb9.3) 1 + 2 R FAP(4B9)- 3.90/95.5/4.5 87 CD28(mAb9.3) 2+2 S FAP (4B9)-CD28 2.63 2.1/96.3/1.6 90.55(mAb9.3) 2+1 T FAP(4B9) - 2.3 0/100/0 100 CD28(mAb9.3) C-02 U FAP(4B9) -23.78 0.68/97.82/1.5 96.1 CD28(mAb9.3) C-05 V CEA-CD28(mAb9.3) 3.10/100/0 100 2 + 2 W CEA-CD28(mAb9.3) 2.25 0/100/0 92.8 1 + 2 Xmonovalent IgG1 20.2 1.4/98.6/0 97.7 CD28 (mAb9.3) Y FAP(4B9)- 1.57 0/100/0 100 CD28(TGN1412)-CEA 1 + 1 + 1

Example 2

Binding and Kinetic Analysis of Bispecific Antibodies of BispecificAntigen Binding Molecules Targeting CD28 and Fibroblast ActivationProtein (FAP) or Carcinoembryonic Antigen (CEA)

2.1 Binding of Bispecific Antibodies Targeting CD28 and FibroblastActivation Protein (FAP) to FAP- or CEA- and to CD28-Expressing Cells

The binding of bispecific FAP-CD28 molecules was tested using humanfibroblast activating protein (huFAP) expressing 3T3-huFAP cells (clone19). This cell line was generated by the transfection of the mouseembryonic fibroblast NIH/3T3 cell line (ATCC CRL-1658) with theexpression vector pETR4921 to express huFAP under 1.511 g/mL Puromycinselection. The binding to human CD28 was tested with CHO cellsexpressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61, modifiedto stably overexpress human CD28). The binding to human CEACAM5 wastested with CEA-expressing MKN45 cells (gastric cancer cell line, DSMZ#ACC 409).

To assess binding, cells were harvested, counted, checked for viabilityand re-suspended at 2.5E5/ml in FACS buffer (eBioscience, Cat No00-4222-26). 5×10⁴ cells were incubated in round-bottom 96-well platesfor 2 h at 4° C. with increasing concentrations of the FAP-targeted CD28constructs (1 pM-100 nM). Then, cells were washed three times with coldFACS buffer, incubated for further 60 min at 4° C. with PE-conjugated,goat-anti human PE (Jackson ImmunoReserach, Cat No 109-116-098), washedonce with cold FACS buffer, centrifuged and resuspended in 100 μl FACSbuffer. To monitor unspecific binding interactions between constructsand cells, an anti-DP47 IgG was included as negative control. Bindingwas assessed by flow cytometry with a FACS Fortessa (BD, Software FACSDiva). Binding curves were obtained using GraphPadPrism6.

The FAP-CD28 molecules were able to bind to both human FAP as well ashuman CD28 on cells in a concentration dependent manner (FIGS. 2B and 2Cfor certain examples). As expected, no binding was detected with theanti-DP47 IgG, indicating that the detection of binding is due tospecific CD28 and FAP binding by the respective targeting moieties.

CEA-CD28 molecules were also able to bind to both human CEA as well ashuman CD28 on cells.

2.2 Kinetic Analysis of Bispecific or Trispecific Antibodies TargetingCD28 and CEA

Affinity (K_(D)) of both binding moieties of the bispecific ortrispecific antibodies comprising anti-CEA (Medi-565) and anti-CD28 wasmeasured by SPR using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated huCD28-Fc antigen and biotinylated Hu N(A2-B2)A-avi-Hisimmobilized on an NLC chip by neutravidin capture.

For the generation of a CEACAM5-based antigen that contains the epitopefor CEA(Medi-565), a chimeric protein consisting of two CEACAM1 and twoCEACAM5 Ig domains was generated. Based on the sequence of CEACAM1, thesecond and third domain of CEACAM1 was replaced by the CEACAM5 domainsA2 and B2. A C-terminal avi-tag and His tag were fused for site-specificbiotinylation and purification. The resulting protein was named HuN(A2-B2)A-avi-His (SEQ ID NO: 169).

Immobilization of recombinant antigens (ligand): Antigens were dilutedwith PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween20) to 10 μg/ml, then injected at 30 μl/minute at varying contact times,to achieve immobilization levels of about 400, 800, and 1600 responseunits (RU) in vertical orientation. Injection of analytes: For one-shotkinetics measurements, injection direction was changed to horizontalorientation, two-fold dilution series of the purified bispecificCEA-targeted anti-CD28 bispecific antibody (varying concentration rangesbetween 50 and 3.125 nM) were injected simultaneously at 50 μl/min alongseparate channels 1-5, with association times of 150 s, and dissociationtimes of 450 s. Buffer (PBST) was injected along the sixth channel toprovide an “in-line” blank for referencing. Association rate constants(kon) and dissociation rate constants (koff) were calculated using asimple one-to-one Langmuir binding model in ProteOn Manager v3.1software by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) wascalculated as the ratio koff/kon. Calculated K_(D) values of abispecific antibody comprising one anti-CD28 antigen binding domain andone anti-CEA antigen binding domain (Molecule M) are in line with themeasured values of the respective monospecific constructs. The kineticand thermodynamic data are summarized in Table 2 below.

TABLE 2 kinetic and thermodynamic analysis of CEA-CD28(SA) 1 + 1(Molecule M) Binding moiety k_(on) (1/(s*M) K_(off) (1/s) K_(D) (nM)Anti-CEA 4.13 exp5 1.2 exp−4 0.29 Anti-CD28 (TGN1412) 3.13 exp5 3.76exp−4 1.2

Example 3

Generation and Characterization of CD28 (SA) Variants Devoid of Hotspotsand Reduced in Affinity

3.1 Removal of an Unpaired Cysteine Residue, Tryptophan Residues, aDeamidation Site and Generation of Affinity-Reduced CD28 (SA) Variants

As part of our detailed binder characterization, a computationalanalysis of the CD28(SA) variable domain sequences was performed. Thisanalysis revealed an unpaired cysteine in the CDR2 region of VH(position 50, Kabat numbering), tryptophan residues in CDR3 of VH(position 100a, Kabat numbering) and CDR1 of VL (position 32, Kabatnumbering), and a potential asparagine deamidation site in CDR2 of VH(position 56, Kabat numbering). While oxidation of tryptophan is arather slow process and can be prevented by adding reducing compounds,the presence of unpaired cysteines in an antibody variable domain can becritical. Free cysteines are reactive and can form stable bonds withother unpaired cysteines of other proteins or components of the cell ormedia. As a consequence, this can lead to a heterogeneous and instableproduct with unknown modifications which are potentially immunogenic andtherefore may pose a risk for the patients. In addition, deamidation ofasparagine and the resulting formation of iso-aspartate and succinimidecan affect both in vitro stability and in vivo biological functions. Acrystal structure analysis of the parental murine binder 5.11A revealedthat C50 is not involved in binding to human CD28 and therefore can bereplaced by a similar amino acid such as serine without affecting theaffinity to CD28 (Table 6, variant 29). Both tryptophan residues as wellas asparagine at position 50, however, are close to or involved in thebinding interface and a replacement by a similar amino acid cantherefore lead to a reduction of the binding affinity. In this example,we particularly aimed at reducing the affinity of CD28(SA) to human CD28because of the following reason: The affinity of CD28(SA) is in therange of 1-2 nM with a binding half-life of about 32 minutes. Thisstrong affinity can lead to a sink effect in tissue containing largeamounts of CD28-expressing cells such as blood and lymphatic tissue wheninjected intravenously into patients. As a consequence, site-specifictargeting of the compound via the targeting component(s) FAP and/or CEAmay be reduced and the efficacy of the construct can be diminished. Inorder to minimize such an effect, several VH and VL variants weregenerated in order to reduce to affinities to different degrees (FIGS.3A and 3C). Besides the previously mentioned positions that representpotential stability hotspots, additional residues involved directly orindirectly in the binding to human CD28 were replaced either by theoriginal murine germline amino acid or by a similar amino acid. Inaddition, the CDRs of both CD28(SA) VL and VH were also grafted into therespective framework sequences of trastuzumab (FIGS. 3B and 3D). Severalcombinations of VH and VL variants were then expressed as monovalentone-armed anti-CD28 IgG-like constructs and binding was characterized bySPR.

3.2 Analysis of the Dissociation Rate Constants (K_(off)) of ReducedOne-Armed Anti-CD28 Variants by SPR

In order to characterize the anti-CD28 binder variants in a first step,all binders were expressed as monovalent one-armed IgG-like constructs(FIG. 4A). This format was chosen in order to characterize the bindingto CD28 in a 1:1 model. 5 days after transfection into HEK cells, thesupernatant was harvested and the titer of the expressed constructs wasdetermined.

The off-rate of the anti-CD28 binder variants was determined by surfaceplasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25°C. with biotinylated huCD28-Fc antigen immobilized on NLC chips byneutravidin capture. For the immobilization of recombinant antigen(ligand), huCD28-Fc was diluted with PBST (Phophate buffered saline withTween 20 consisting of 10 mM phosphate, 150 mM sodium chloride pH 7.4,0.005% Tween 20) to concentrations ranging from 100 to 500 nM, theninjected at 25 μl/minute at varying contact times. This resulted inimmobilization levels between 1000 to 3000 response units (RU) invertical orientation.

For one-shot kinetics measurements, injection direction was changed tohorizontal orientation. Based on the titer of the produced supernatants,the monovalent one-armed IgGs were diluted with PBST to get two-folddilution series ranging from 100 nM to 6.25 nM. Injection was performedsimultaneously at 50 μl/min along separate channels 1-5, withassociation times of 120 s, and dissociation times of 300 s. Buffer(PBST) was injected along the sixth channel to provide an “in-line”blank for referencing. Since the binding interaction was measured withmonovalent one-armed IgGs from supernatant without purification andbiochemical characterization, only the off-rates of the protein:proteininteraction was used for further conclusions. Off-rates were calculatedusing a simple one-to-one Langmuir binding model in ProteOn Manager v3.1software by fitting the dissociation sensorgrams. The dissociation rateconstants (k_(off)) values of all clones are summarized in Table 3.Comparison of the produced variants revealed k_(off) values with an upto 30-fold decrease compared to the parental sequence.

TABLE 3 Summary of all expressed monovalent anti-CD28 variants withdissociation rate constants (k_(off)) values SEQ SEQ SEQ Tapir ID ID IDBinder variants ID NO: NO: NO: K_(off)(10⁻⁴/M) CD28(SA)_variant_1P1AE4441 112 65 126 3.0 (parental CD28) CD28(SA)_variant_2 P1AE3058 113120 126 N/A CD28(SA)_variant_3 P1AE3059 113 121 126 N/ACD28(SA)_variant_4 P1AE3060 113 122 126 N/A CD28(SA)_variant_5 P1AE3061113 65 126 N/A CD28(SA)_variant_6 P1AE3062 114 120 126 N/ACD28(SA)_variant_7 P1AE3063 114 121 126 100 CD28(SA)_variant_8 P1AE3064114 122 126 68 CD28(SA)_variant_9 P1AE3065 114 123 126 78CD28(SA)_variant_10 P1AE3066 114 124 126 N/A CD28(SA)_variant_11P1AE3067 114 65 126 37 CD28(SA)_variant_12 P1AE3068 115 125 126 2.4CD28(SA)_variant_13 P1AE3069 115 65 126 1.9 CD28(SA)_variant_14 P1AE3070116 120 126 100 CD28(SA)_variant_15 P1AE3071 116 121 126 24CD28(SA)_variant_16 P1AE3072 116 122 126 10 CD28(SA)_variant_17 P1AE3073116 123 126 14 CD28(SA)_variant_18 P1AE3074 116 124 126 82CD28(SA)_variant_19 P1AE3075 116 65 126 2.9 CD28(SA)_variant_20 P1AE3076117 120 126 N/A CD28(SA)_variant_21 P1AE3077 117 121 126 N/ACD28(SA)_variant_22 P1AE3078 117 122 126 61 CD28(SA)_variant_23 P1AE3079117 65 126 43 CD28(SA)_variant_24 P1AE3080 118 120 126 80CD28(SA)_variant_25 P1AE3081 118 121 126 3.51 CD28(SA)_variant_26P1AE3082 118 122 126 9.7 CD28(SA)_variant_27 P1AE3083 118 123 126 14CD28(SA)_variant_28 P1AE3084 118 124 126 69 CD28(SA)_variant_29 P1AE3085118 65 126 2.5 CD28(SA)_variant_30 P1AE3086 119 125 126 3.22CD28(SA)_variant_31 P1AE3087 119 65 126 2.5

Binding to human CD28 was tested with CHO cells expressing human CD28(parental cell line CHO-k1 ATCC #CCL-61. This binding assay is describedin Example 4 below. The monovalent one-armed IgG-like CD28 variantconstructs showed differences in binding as can be seen from FIGS. 4A to4C.

3.3 Preparation and Kinetic Analysis of Bispecific FAP-TargetedAnti-CD28 Affinity Variants

Based on the off-rate analysis and the binding study on CD28-expressingcells, several combinations of anti-CD28 VH and VL variants withdifferent binding intensities were selected and expressed asFAP-targeted bispecific huIgG1 PG-LALA CrossFab molecules (forcombinations of SEQ ID NOs: see Table 4). The resulting constructs in1+1 format (FIG. 4B) were purified and a biochemical analysis wasperformed (Table 5).

TABLE 4 Summary of all expressed 1 + 1 bispecific FAP-targeted anti-CD28variants SEQ ID SEQ ID SEQ ID SEQ ID Binder variants Tapir ID NO: NO:NO: NO: FAP (4B9)-CD28 P1AE3131 67 68 114 122 (CD28(SA)_Variant 8) 1 + 1FAP (4B9) - CD28 P1AE3132 67 68 114 65 (CD28(SA)_Variant 11) 1 + 1 FAP(4B9) - CD28 P1AB3133 67 68 115 125 (CD28(SA)_Variant 12) 1 + 1 FAP(4B9) - CD28 P1AE3134 67 68 116 121 (CD28(SA)_Variant 15) 1 + 1 FAP(4B9) - CD28 P1AE3135 67 68 116 122 (CD28(SA)_Variant 16) 1 + 1 FAP(4B9) - CD28 P1AB3136 67 68 116 123 (CD28(SA)_Variant 17) 1 + 1 FAP(4B9) - CD28 P1AE3137 67 68 116 65 (CD28(SA)_Variant 19) 1 + 1 FAP(4B9) - CD28 P1AE3138 67 68 117 65 (CD28(SA)_Variant 23) 1 + 1 FAP(4B9) - CD28 P1AE3139 67 68 118 121 (CD28(SA)_Variant 25) 1 + 1 FAP(4B9) - CD28 P1AE3140 67 68 118 123 (CD28(SA)_Variant 27) 1 + 1 FAP(4B9) - CD28 P1AE3141 67 68 118 65 (CD28(SA)_Variant 29) 1 + 1

TABLE 5 Summary of the production and purification of FAP-targeted anti-CD28 variants Analytical SEC Purity Yield (HMW/Monomer/LMW) measured byTaPIR ID Bispecific molecules [mg/l] [%] CE-SDS [%] P1AE3131 FAP(4B9)-CD28 11.8 0.1/98.5/1.4 100 (CD28(SA)_Variant 8) 1 + 1 P1AE3132 FAP(4B9) - CD28 8.1 0.5/97.4/2.1 100 (CD28(SA)_Variant 11) 1 + 1 P1AE3133FAP (4B9) - CD28 6.1 0/100/0′ 100 (CD28(SA)_Variant 12) 1 + 1 P1AE3134FAP (4B9) - CD28 9.2 0/100/0 100 (CD28(SA)_Variant 15) 1 + 1 P1AE3135FAP (4B9) - CD28 0.4 0/100/0 97 (CD28(SA)_Variant 16) 1 + 1 P1AE3136 FAP(4B9) - CD28 1.35 0/78.7/21.3 87 (CD28(SA)_Variant 17) 1 + 1 P1AE3137FAP (4B9) - CD28 2.6 0/100/0 100 (CD28(SA)_Variant 19) 1 + 1 P1AE3138FAP (4B9) - CD28 15.5 0/97.5/2.5 98 (CD28(SA)_Variant 23) 1 + 1 P1AE3139FAP (4B9) - CD28 5.4 0/88.7/11.3 100 (CD28(SA)_Variant 25) 1 + 1P1AE3140 FAP (4B9) - CD28 9.7 0/98.3/1.7 96 (CD28(SA)_Variant 27) 1 + 1P1AE3141 FAP (4B9) - CD28 1.76 1/99/0 96.3 (CD28(SA)_Variant 29) 1 + 1

Affinity (K_(D)) of the produced bispecific antigen binding molecules toCD28 was measured by SPR using a ProteOn XPR36 instrument (Biorad) at25° C. with biotinylated huCD28-Fc antigen immobilized on NLC chips byneutravidin capture. Immobilization of recombinant antigens (ligand):Antigen was diluted with PBST (10 mM phosphate, 150 mM sodium chloridepH 7.4, 0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute atvarying contact times, to achieve immobilization levels of about 200,400 or 800 response units (RU) in vertical orientation. Injection ofanalytes: For one-shot kinetics measurements, injection direction waschanged to horizontal orientation, two-fold dilution series of purifiedbispecific FAP-targeted anti-CD28 affinity variants (varyingconcentration ranges between 50 and 3.125 nM) were injectedsimultaneously at 50 μl/min along separate channels 1-5, withassociation times of 150 s, and dissociation times of 450 s. Buffer(PBST) was injected along the sixth channel to provide an “in-line”blank for referencing. Association rate constants (k_(on)) anddissociation rate constants (k_(off)) were calculated using a simpleone-to-one Langmuir binding model in ProteOn Manager v3.1 software bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) was calculated as the ratiok_(off)/k_(on). Analyzed clones revealed K_(D) values in a broad range(between 1 and 25 nM). The kinetic and thermodynamic data are summarizedin Table 6.

TABLE 6 kinetic and thermodynamic analysis of expressed FAP-targetedanti-CD28 variants Bispecific molecule k_(on) (1/(s*M) k_(off) (1/s)K_(D) (nM) parental 3.79 exp5  3.6 exp−4 1 FAP (4B9)-CD28 2.19 exp5 5.21exp−3 23.8 (CD28(SA)_Variant 8) 1 + 1 FAP (4B9) - CD28  2.3 exp5 2.87exp−3 12.5 (CD28(SA)_Variant 11) 1 + 1 FAP (4B9) - CD28 2.6 1exp5 2.67exp−4 1 (CD28(SA)_Variant 12) 1 + 1 FAP (4B9) - CD28 2.59 exp5 1.84exp−3 7.1 (CD28(SA)_Variant 15) 1 + 1 FAP (4B9) - CD28 1.87 exp5 9.94exp−4 5.3 (CD28(SA)_Variant 16) 1 + 1 FAP (4B9) - CD28 3.38 exp5 1.25exp−3 3.7 (CD28(SA)_Variant 17) 1 + 1 FAP (4B9) - CD28  2.8 exp5 3.04exp−4 1.1 (CD28(SA)_Variant 19) 1 + 1 FAP (4B9) - CD28 2.11 exp5 3.42exp−3 16.3 (CD28(SA)_Variant 23) 1 + 1 FAP (4B9) - CD28 2.38 exp5 3.96exp−4 1.7 (CD28(SA)_Variant 25) 1 + 1 FAP (4B9) - CD28 2.27 exp5 1.21exp−3 5.4 (CD28(SA)_Variant 27) 1 + 1 FAP (4B9) - CD28 2.72 exp5 3.07exp−4 1.1 (CD28(SA)_Variant 29) 1 + 1

Example 4

Binding of Monovalent CD28 Agonistic IgGs and FAP-Targeted CD28Agonistic Antibodies to CD28-Expressing and FAP-Expressing Cells

Binding to human CD28 was tested with CHO cells expressing human CD28(parental cell line CHO-k1 ATCC #CCL-61, modified to stably overexpresshuman CD28). To assess binding, cells were harvested, counted, checkedfor viability and re-suspended at 2.5×10⁵/ml in FACS buffer(eBioscience, Cat No 00-4222-26). 5×10⁴ cells were incubated inround-bottom 96-well plates for 2 h at 4° C. with increasingconcentrations of the CD28 binders (1 pM-100 nM). Then, cells werewashed three times with cold FACS buffer, incubated for further 60 minat 4° C. with PE-conjugated, goat-anti human PE (Jackson ImmunoReserach,Cat No 109-116-098), washed once with cold FACS buffer, centrifuged andresuspended in 100 ul FACS buffer. To monitor unspecific bindinginteractions between constructs and cells, an anti-DP47 IgG was includedas negative control. Binding was assessed by flow cytometry with a FACSFortessa (BD, Software FACS Diva). Binding curves were obtained usingGraphPadPrism6.

The monovalent one-armed IgG-like CD28 variant constructs showeddifferences in binding as can be seen from FIGS. 4A to 4C. Furthermore,the binding of bispecific FAP-targeted anti-CD28 antibodies in 1+1format to CHO cells expressing human CD28 was determined. The K_(D)values for the different 1+1 constructs with selected CD28 variants areshown in Table 7 below or in the corresponding graphs of FIGS. 4D and4E.

TABLE 7 Binding of FAP-targeted anti-CD28 1 + 1 constructs to CHO cellsexpressing human CD28 Binder TAPIR K_(D) (nM) TGN1412 P1AD4492 1 variant8 P1AE3131 23.8 variant 11 P1AE3132 12.5 variant 12 P1AE3133 1 variant15 P1AE3134 7.1 variant 16 P1AE3135 5.3 variant 17 P1AE3136 3.7 variant19 P1AE3137 1.1 variant 23 P1AE3138 16.3 variant 25 P1AE3139 1.7 variant27 P1AE3140 5.4 variant 29 P1AE3141 1.1

The binding of bispecific FAP-targeted anti-CD28 antibodies in 1+1format to FAP-expressing 3T3-huFAP cells (clone 19) was also determinedas described in Example 2.1 and is shown in the corresponding graphs ofFIGS. 4F and 4G.

Example 5

Binding of CEA-Targeted CD28 Agonistic Antibodies to CD28-ExpressingCells

Binding to human CD28 was tested with CHO cells expressing human CD28(Binding was assessed by flow cytometry with a FACS Fortessa (BD,Software FACS Diva) as described in Example 4. Binding curves wereobtained using GraphPadPrism6. The binding curves for the different 1+1constructs with selected CD28 variants are shown in FIG. 16 .

Example 6

In Vitro Functional Characterization of Targeting CD28 and FibroblastActivation Protein (FAP) or Carcinoembryonic Antigen (CEA)

Several cell-based in vitro assays were performed with primary humanPBMCs to evaluate the activity of CD28(SA) and bispecific FAP-targetedCD28 antigen binding molecules in the presence and absence of TCRsignals provided by T-cell bispecific-(TCB) antibodies. T-cellproliferation, cytokine secretion, and tumor cell killing as determinedby flow cytometry, cytokine ELISA, and live cell imaging were obtainedas read-outs.

-   -   1. The activity of the original superagonistic CD28(SA) IgG4 was        assessed using a previously described high density pre-culture        system to restore the responsiveness of peripheral blood derived        T cells towards CD28-mediated superagonism (Römer et al., 2011).    -   2. The functionality of targeted CD28 molecules in absence of        TCR signals was assessed in a primary human PBMC co-culture        assay, wherein FAP- or CEA-targeted CD28 molecules were        crosslinked by simultaneous binding to human CD28 on T cells and        human FAP, expressed on either 3T3-huFAP cells (parental cell        line ATCC #CCL-92, modified to stably overexpress human FAP) or        MCSP- and FAP-expressing MV3 melanoma cells, or CEA-expressing        MKN45 gastric cancer cells, respectively.    -   3. The functionality of FAP-targeted CD28 molecules in presence        of TCR signals was assessed as described above, with the        additional presence of a TCB molecule, crosslinked by        simultaneous binding to CD3 on T cells and, either human CEA on        MKN45 gastric cancer cells, Lovo colon cancer cells, HT-29 colon        cancer cells, or MCSP, expressed on MV3 melanoma cells.

PBMC Isolation

Peripheral blood mononuclear cells (PBMCs) were prepared by densitygradient centrifugation from enriched lymphocyte preparations ofheparinized blood obtained from a Buffy Coat (Blutspende Zurich). 25 mlof blood (diluted 1:2 in PBS) were layered over 15 ml lymphoprep(STEMCELL technologies, Cat No 07851) and centrifuged at roomtemperature for min at 845 xg without brake. The PBMC-containinginterphase was collected in 50 ml tubes with a 10 ml pipette. The cellswere washed with PBS and centrifuged 5 min at 611 xg. The supernatantwas discarded, the pellet re-suspended in 50 ml PBS and centrifuged for5 min at 304 xg. The washing step was repeated, centrifuging at 171 xg.The cells were re-suspended in RPMI 1640 Glutamax (containing 5% humanserum, sodium pyruvate, NEAA, 50 μM 2-mercaptoethanol,Penicillin/Streptomycin) and processed for further functional analysisaccording to the respective assay protocol.

High Density Pre-Culture of PBMCs and In Vitro Assessment of T CellActivation by the CD28 Superagonist CD28(SA)

To restore the responsiveness of human T cells to TGN1412-mediated CD28superagonism, PBMCs were pre-cultured at high density (HD) (Römer et al,2011) before assessing the effects of CD28 superagonistic antibodies. Inbrief, PBMCs were adjusted to 1E7 cells/ml in complete medium (RPMI 1640Glutamax, 5% human serum, Sodium-Pyruvate, NEAA, 50 μM2-Mercaptoethanol, Penicillin/Streptomycin) and cultured at 1.5 ml/wellin a 24-well plate for 48 hours at 37° C., 5% CO₂. Cells were thenre-harvested, washed in complete medium, centrifuged at 550 xg for 5 minand adjusted with to the desired cell density required for functionalcharacterization. To assess T cell proliferation, PBMCs were labelledwith CFSE and CFSE-dilution was measured as proxy for T cellproliferation after 5 days of stimulation. In brief, cells were adjustedto 2×10⁷/ml in PBS and labelled with 2.5 μM CFSE proliferation dye(LifeTechnologies, Cat No 65-0850-84) for 6 minutes at 37° C., 5% CO₂.Cells were washed once in complete medium, followed by 2 washing stepsin PBS. For stimulation with TGN1412, PBMCs were adjusted to 2×10⁶/ml incomplete medium and 1×10⁵ cells were distributed to each well of a flatbottom 96-well plate and stimulated with increasing concentrations ofTGN1412 (0.0002 nM to 10 nM, triplicates). CF SE-dilution was assessedby flow cytometry. Briefly, cells were centrifuged at 550 xg for 5 minand washed with PBS. CF SE-dilution was assessed by flow cytometry.Briefly, cells were centrifuged at 550 xg for 5 min and washed with PBS.Surface staining for CD8 (BV711 anti-human CD8a, BioLegend #301044), CD4(PE-Cy7 anti-human CD4, BioLegend #344612) was performed according tothe suppliers' indications. Cells were then washed twice with 150μl/well PBS and resuspended in 200 μl/well FACS buffer and analyzedusing BD FACS Fortessa. Cytokine secretion was measured at day 5 postactivation via cytokine ELISA (huTNFα, DuoSet #DY210-05 and huIFNγ,DuoSet #DY285-05) or cytokine multiplex (Human Cytokine 17-plex assay,Bio-Rad #M5000031YV) analysis from culture supernatants.

In Vitro Assessment of T Cell Proliferation and Cytokine Secretion byBispecific FAP-Targeted CD28 Antigen Binding Molecules in Absence andPresence of TCB Signals

Pan T cells were used as effector cells and isolated from PBMCs by MACS,using the Pan T Cell Isolation Kit (Miltenyi Biotec) according to themanufacturer's instructions.

To measure T cell activation by bispecific FAP-CD28 antigen bindingmolecules in absence of TCB, CFSE-labelled pan T cells were co-culturedwith 3×10⁴/well 3T3-huFAP or parental 3T3 cells lacking FAP expression(3T3-WT), seeded the previous day in flat-bottom 96-well plates.Bispecific FAP-CD28 antigen binding molecules were added in increasingconcentrations (0.0002 nM-10 nM, triplicates).

To measure T cell proliferation in presence of a TCB signal,CFSE-labelled pan T cells were incubated with 3×10⁴ FAP- andMCSP-expressing MV3 cells/well, seeded the previous day in flat-bottom96-well plates, increasing concentrations of bispecific FAP-CD28 antigenbinding molecules (0.0002 nM-10 nM, triplicates), and fixedconcentration of MCSP-TCB (5 pM, P1AD2189). As controls, wellscontaining only TCB were included.

CFSE-dilution was assessed by flow cytometry and cytokine secretion wasmeasured at 5 days post activation via cytokine ELISA (huTNFα, DuoSet#DY210-05 and huIFNγ, DuoSet #DY285-05) or cytokine multiplex (HumanCytokine 17-plex assay, Bio-Rad #M5000031YV) analysis from culturesupernatants.

The preparation of the anti-MCSP/anti-CD3 bispecific antibody (MCSP-TCB)used in the experiment is described in WO 2014/131712 A1.

Superagonism of CD28(SA) Requires FcγRIIb Cross-Linking

High Density Pre-Culture of PBMCs Restores CD28(SA) Superagonism

To understand the mechanism of action of CD28(SA), we validated highdensity (HD) pre-culture of PBMCs as a previously described protocol torestore the ability of PBMC-derived T cells to respond toTGN1412-mediated CD28 superagonism (Römer et al., 2011). As depicted inFIGS. 5A and 5B, CD28(SA) IgG4 (P1AE1975) induces PBMC T cellproliferation (FIG. 5A) and cytokine production (FIG. 5B) in aconcentration-dependent manner at 5 days post stimulation only in PBMCssubjected to HD pre-culture, while fresh PBMCs remained unresponsive. Weconcluded that the previously published protocol to restore T cells'responsiveness to CD28(SA) in vitro (Römer et al., 2011) could bereproduced in our hands.

CD28(SA) Superagonistic Activity Requires Cross-Linking ViaFcγRIIb—Blocking of FcγRIIb Abolishes CD28(SA) Functionality

Previously published literature indicates that TGN1412 potentiallyrelies on FcγRIIb cross-linking. To understand the link between HDpre-culture of PBMCs and Fc-dependence of CD28(SA) functionality, theexpression levels of FcγRIIb on PBMCs were assessed by flow cytometrybefore and after HD pre-culture. As depicted in FIG. 5C, FcγRIIbexpression was absent in fresh PBMC monocytes, while 96.8% of monocytesexpressed FcγRIIb after 2 days of HD pre-culture. Antibody-mediatedblocking of FcγRIIb in subsequent T cell proliferation assays completelyabrogated T cell proliferation upon stimulation with CD28(SA), measuredafter 5 days in culture (FIG. 5D). In an alternative approach, anFc-silenced variant of CD28(SA) which carries the P329G-LALA mutation(CD28(SA) IgG1 PG-LALA: P1AD9289) did not display superagonisticfunction (FIG. 6A). These data confirm that CD28(SA)-mediated CD28superagonism relies on cross-linking via FcγRIIb.

Adding a Tumor-Targeting Moiety for FAP-Targeting to Fc-Silent CD28(SA)Restores Superagonism, which is then Dependent on the Presence of theTumor Target

Given that CD28 superagonism by TGN1412 relies on FcγRIIb cross-linking,we hypothesized that FcR-dependence may be re-directed to tumors byintroduction of (i) an Fc-silencing P329G-LALA mutation and (ii) atargeting moiety that cross-links to a surface-expressed tumor-antigen.To test this hypothesis, a FAP-targeting moiety was added as C-terminalfusion to an Fc-silenced TGN1412 (FAP-CD28 1+2 SA: P1AD9011). SinceFcR-crosslinking was not required for this approach, PBMCs were notsubjected to HD pre-culture. Instead, fresh PBMCs were co-cultured with3T3-huFAP or 3T3-WT for 5 days in presence of increasing concentrationof FAP-CD28 (P1AD9011) and T cell proliferation was assessed byCFSE-dilution via flow cytometry. As shown in FIG. 6B, the introductionof FAP-binding moiety enabled T cell proliferation exclusively in thepresence of FAP. We concluded that superagonism can be selectivelytargeted to tumor antigens by Fc-silencing and addition of atumor-targeting moiety.

Conventional CD28 Agonistic Antibodies (Clone 9.3) do not BehaveSuperagonistically in Tumor-Targeted Bispecific Formats

Two types of CD28 agonistic antibodies have been reported in theliterature: superagonistic CD28 antibodies such as TGN1412 are able toautonomously activate T cells without the necessity of an additionalsignal provided by TCR. These antibodies are referred to assuperagonists, because they surpass the functionality of natural CD28agonistic ligands CD80 and CD86, which strictly rely on the presence ofa TCR signal to enhance T cell function. In contrast to superagonisticantibodies such as TGN1412, conventional agonistic antibodies such asclone mab 9.3 are not able to activate T cells autonomously, but, justlike the natural CD28 ligands, require an additional TCR signal toenhance T cell activity. To assess the effect of targeting CD28 agoniststo tumor antigens in more detail, we generated further FAP-CD28molecules: (i) a superagonistic (SA) molecule with 2 CD28 bindingmoieties (TGN1412) and 2 FAP binding moieties=2+2 SA format (P1 AD4493),(ii) a conventional agonist (CA) with 2 CD28 binding moieties (clone9.3) and 1 or 2 FAP binding moieties, respectively: 2+2 CA (P1AD8968),1+2 CA (P1 AD8962). Fresh PBMCs were co-cultured with 3T3-huFAP or3T3-WT for 5 days in presence of increasing concentration of theFAP-targeted molecules and T cell proliferation was assessed by CFSE-dilution via flow cytometry. As depicted in FIGS. 7A to 7D, onlysuperagonistic binders were able to activate T cells. Further, T cellactivation via the described superagonistic constructs is strictlydependent on the presence of FAP (FIG. 7B), as demonstrated by absent Tcell activation in absence of FAP (FIG. 7D). In line with these data,also cytokine secretion was only observed for constructs harboring thesuperagonistic CD28(SA) antibodies, but not the conventional agonistic9.3 antibody (FIG. 7E). We concluded that only superagonistic CD28antibodies elicit autonomous T cell activation in bispecifictumor-targeted antibody formats, while the same formats withconventional 9.3 binders are not superagonistic.

Example 7

In Vitro Assessment of Tumor Cell Killing by Tumor-Targeted CD28Molecules in the Absence or Presence of TCB

To assess the ability of bispecific FAP-CD28 or CEA-CD28 antigen bindingmolecules to achieve tumor cell killing or support TCB-mediated tumorcell killing, purified pan T cells served as effector cells andRFP-expressing MV3 cells and MKN45 cells, respectively, served as tumortargets.

To assess killing of MV3 tumor cells, 5000 MV3 target cells seeded theprevious day were co-cultured with 1×10⁵ pan T cells per well in flatbottom 96-well plates (E:T 20:1), in presence of 5 pM MCSP-TCB(P1AD2189) alone or in combination with 10 nM bispecific FAP-CD28antigen binding molecule. To assess killing of MV3 tumor cells, 5000 MV3target cells seeded the previous day were co-cultured with 1×10⁵ pan Tcells per well in flat bottom 96-well plates (E:T 20:1), in presence of2 nM FAP-CD28. To assess the killing of MKN45 tumor cells, 5000 MKN45,seeded the previous day, were co-cultured with 1×10⁵ pan T cells perwell in flat-bottom 96-well plates in presence of 2 nM CEA-CD28. Killingof target cells was monitored over the course of 90 hours, using theIncuCyte live cell imaging system (Essen Biosciences), capturing 4images per well every 3 hours. RFP+ object counts per image (assessedvia IncuCyte ZOOM software, Essen Biosciences) over time served as proxyfor target cell death. Antibody-mediated target cell killing wasdistinguished from spontaneous target cell death by monitoring counts oftarget cells in presence of effector T cells alone over time (=baselinecontrol). Killing was calculated as 100−x, x being % targets relative tothe baseline control. Statistical analyses were performed usingstudent's t-test, comparing the areas under the curves (AUC) of %killing over time.

FAP-CD28 Induces Target Cell Killing in the 1+2 Format, but Only withSuperagonistic CD28 Binders, not with Conventional CD28 AgonisticBinders

The ability of FAP-CD28 molecules to induce tumor cell killing wasassessed. As depicted in FIGS. 8A to 8D, co-culture of PBMC-derived Tcells with FAP-expressing MV3 melanoma cells in presence of FAP-CD28over 90 hours led to killing of MV3 cells exclusively by FAP CD28(SA) in1+2 format (P1AD9011) and was comparable to the induction of killingachieved by a FAP-targeted TCB (7). No killing was observed withFAP-CD28(SA) in 2+2 format (P1AD4493) as well as FAP-CD28 withconventional CD28 agonistic 9.3 antibody (P1AD8968 & P1AD8962). Weconclude that in addition to T cell proliferation and cytokinesecretion, a FAP-CD28 in 1+2 format with superagonistic binders can alsoelicit target cell killing, comparable to a TCB.

CEA-CD28 Induces Target Cell Killing in the 1+2 and 2+2 Format, but Onlywith Superagonistic Antibodies, not with Conventional CD28 AgonisticAntibodies

In an alternative approach, we used CEA-targeted CD28 agonisticmolecules in the 2+2 SA (P1AE1195), 1+2 SA (P1AE1194), 2+2 CA(P1AE1193), and 2+1 CA (P1AE1192) formats to assess their ability toinduce target cell killing. PBMC T cells were co-cultured withCEA-expressing MKN45 cells in presence of CEA-CD28 in the aforementionedformats for 90 h. Both formats containing superagonistic CD28 binderswere able to induce killing of CEA-expressing MKN45 cells (FIGS. 9A and9B). We speculate that the discrepancy between FAP-CD28(SA) 2+2 andCEA-CD28 (SA) 2+2's ability to kill their respective target cells lieswithin discrepancies of target expression levels in MKN45 vs. MV3 cells.Precisely, in house data confirmed that FAP-expression levels of MV3cells are 10× lower than CEA-expression levels of MKN45 cells. Thus, inMV3 cells, tumor target binding sites might be limiting and killing ofMV3 cells requires efficient occupancy of FAP vs. CD28, which isadvantageous in the 1+2 format (i.e. 1 FAP binding site cross-links 2CD28 binding sites) compared to the 2+2 (i.e. 2 FAP binding sitesrequired for cross-linking of 2 CD28 binding sites).

CD28 Superagonism by TGN1412 Binders Relies on CD28 BinderMultivalency—Monovalent Binders are not Superagonistic

To further investigate the nature of CD28 superagonism, we assessed ifmonovalent CD28 TGN1412 binders display superagonistic behavior in atumor-targeted bispecific format. PBMC T cells were co-cultured with3T3-huFAP cells and incubated with increasing concentrations FAP-CD281+2 SA with CD28 bivalency (P1AD9011) and FAP-CD28 1+1 SA with CD28monovalency (P1AD4492). As displayed in FIG. 10A, FAP-CD28 withmonovalent CD28 binding (P1AD4492) was not able to induce T cellproliferation, as opposed to the CD28 bivalent construct (P1AD9011).Consistently, upregulation of the T cell activation markers CD69 andCD25 was only observed with the CD28 bivalency (FIGS. 10B and 10C,respectively). In conclusion, TGN1412-mediated superagonism does notonly rely on cross-linking via Fc receptors but also requires CD28binder multivalency.

In conclusion, it could be established that CD28 superagonism can betargeted specifically to tumor antigens by Fc-silencing and introductionof an antigen binding domain capable of specific binding to atumor-associated antigen. Further, tumor-targeted bispecific antibodiesare only superagonistic when they comprised CD28(SA)-based binders andnot when they comprised conventional agonistic binders (clone 9.3).Further, superagonism requires multivalency of the CD28(SA) binder andmonovalent CD28(SA) binding in bispecific constructs abrogatessuperagonistic T cell activation.

FAP-CD28 Supports TCB-Mediated Target Cell Killing and Requires CD28Binder Monovalency to Sustain Tumor Target Dependence

CD28 signaling is well described to enhance T cell receptor mediated Tcell responses. Therefore, T cell bispecific antibodies (TCBs) are apromising combination partner for CD28 agonism. Through combination oftargeted CD28 agonism with TCBs, we envision to enhance TCB-mediatedeffector functions, lower the threshold of CEA-expression for efficientTCB-mediated T cell activation, provide survival cues and supportresistance towards T cell suppression via PD-1 and CTLA4.

To investigate if targeted CD28 agonists can enhance TCBs, we assessedthe ability of FAP-CD28(SA) 1+2 (P1AD9011) and FAP-CD28(SA) 1+1(P1AD4492) to support TCB-mediated target cell killing. Co-culture ofPBMC-derived T cells with MCSP- and FAP co-expressing MV3 cells for 5days in the presence of increasing concentrations of FAP-CD28 and fixed,limiting concentration of MCSP-TCB (5 pM) led to increased killing ofMV3 target cells in a FAP-CD28 concentration-dependent manner (FIG.11A). However, the presence of the TCB abolished FAP-dependence ofFAP-CD28 in the CD28 bivalent format (P1AD9011), while FAP-dependencewas maintained in the CD8 monovalent format (P1AD4492), as demonstratedby a concentration-dependent increase in CEACAM5-TCB-mediated targetcell killing in presence of CEA-expressing, FAP-negative MKN45 tumorcells at 5 days post stimulation (FIG. 11B).

In an alternative approach, we assessed T cell proliferation induced byFAP-CD28 1+2 SA (P1AD9011) in presence or absence of TCB and in presenceor absence of FAP, respectively. As shown in FIG. 12A and in theprevious example, FAP-CD28(SA) 2+1 is strictly dependent on the presenceof FAP for T cell activation in absence of TCBs. In presence of TCBs,however, as shown in FIG. 12B, FAP-CD28(SA) 1+2 induces enhancement of Tcell activation even in absence of FAP.

We hypothesize that the TCB-induced TCR signal potentially leads tosufficient pre-clustering of TCR signaling components, thus renderingsurface cross-linking of CD28 receptors on T cells by bivalent CD28molecules sufficient to elicit co-stimulation. We conclude that for TCBcombination approaches, CD28 binder monovalency is strictly required tomaintain tumor-target dependence of the targeted CD28 agonistic antigenbinding molecule.

Comparison of Various Monovalent FAP-CD28 Formats Reveals a Set ofVarious Functional FAP-CD28 Formats, with Highest Potency for Classical1+1 Format

To assess the impact of the specific antibody format of FAP-CD28 on itsability to enhance TCB-mediated T cell activation, different formats ofFAP-CD28 antigen binding molecules with monovalent CD28 binding weregenerated and are depicted in FIGS. 1C, 1K, 1L and 1M. FAP-CD28 1+2 withCD28 bivalency was used as a reference. To assess the functionality ofthese formats, PBMC T cells were incubated with MCSP- and FAPco-expressing MV3 cells for 5 days in the presence of increasingconcentrations of FAP-CD28 formats together with fixed, limitingconcentration of MCSP-TCB (5 pM). All formats were able to significantlyincrease CD8 T cell proliferation (FIG. 13A), CD4 T cell proliferation(FIG. 13B), and Target cell killing (FIG. 13C). Of note, the potency ofthe molecule C (P1AD4492) was highest, and comparable to the potency ofthe bivalent CD28 reference format 1+2 SA (P1AD9011). Binding to CD28and FAP of all molecules is shown in FIGS. 2F and 2G, respectively.

Example 8

In Vitro Assessment of Tumor Cell Killing of the Combination ofTumor-Targeted CD28 Molecules and CEA-Targeted TCBs

Preparation of T-Cell Bispecific (TCB) Antibodies

TCB molecules have been prepared according to the methods described inWO 2014/131712 A1 or WO 2016/079076 A1. The preparation of theanti-CEA/anti-CD3 bispecific antibody (CEA CD3 TCB or CEA TCB) used inthe experiments is described in Example 3 of WO 2014/131712 A1. CEA CD3TCB is a “2+1 IgG CrossFab” antibody and is comprised of two differentheavy chains and two different light chains. Point mutations in the CH3domain (“knobs into holes”) were introduced to promote the assembly ofthe two different heavy chains. Exchange of the VH and VL domains in theCD3 binding Fab were made in order to promote the correct assembly ofthe two different light chains. 2+1 means that the molecule has twoantigen binding domains specific for CEA and one antigen binding domainspecific for CD3. CEACAM5 CD3 TCB has the same format, but comprisesanother CEA binder and comprises point mutations in the CH and CLdomains of the CD3 binder in order to support correct pairing of thelight chains. CEA CD3 TCB comprises the amino acid sequences of SEQ IDNO:161, SEQ ID NO:162, SEQ ID NO:163 and SEQ ID NO:164. CEACAM5 CD TCBcomprises the amino acid sequences of SEQ ID NO:165, SEQ ID NO:166, SEQID NO:167 and SEQ ID NO:168.

CEA-CD28 Synergizes with CEACAM5-TCB in Target Cell Killing

In an alternative approach, we generated a CEA-CD28(SA) 1+1 bispecificantigen binding molecule (Molecule M, P1AE3127) and assessed its abilityto enhance CEACAM5-TCB mediated target cell killing. To this end,CEA-expressing MKN45 colorectal cancer cells were co-cultured with PBMCT cells and CEA-CD28 (Molecule M, P1AE3127) or untargeted monovalentCD28 (Molecule L, P1AD8944) in presence or absence of suboptimalCEACAM5-TCB (10 nM) and MKN45 cell killing was assessed over time. Asshown in FIG. 14 , only the combination of CEACAM5-TCB and CEA-CD28 ledto target cell killing, while the compounds alone did not achieve targetcell killing, indicating synergistic effects. Further, untargeted CD28in combination with CEACAM5-TCB also did not induce killing,highlighting once more the requirement of monovalent CD28 agonists forcross-linking and the thereby sustained dependence on the tumor target.

CEA-CD28 Enhances CEA-TCB and CEACAM5-TCB and Lowers CEA-ExpressionThresholds on Cancer Cells for TCBs to Activate T Cells

CEA-TCB and CEACAM5-TCB require a certain expression level of CEA ontarget cells to T cell activation and target cell killing. We assessedwhether CEA-CD28 was able to lower the CEA-expression threshold for TCBsto induce efficient target cell killing. To this end, PBMC T cells wereincubated with increasing concentrations of either CEA-TCB (P1AD4646) orCEACAM5-TCB (P1AD5299) and fixed concentrations of CEA-CD28 (Molecule M,P1AE3127) in presence of target cell lines with different CEA expressionlevels: (i) MKN45 (high expression, approx. 400 000 CEA bindingsites/cell), (ii) Lovo (medium expression, approx. 60 000 CEA bindingsites/cell), (iii) HT-29 (low expression, approx. 6 000 CEA bindingsites/cell). T cell proliferation was measured as proxy of T cellactivation. As shown in FIG. 15 , CEA-CD28 could significantly increasethe potency of CEA-TCB and CEACAM5-TCB. Most strikingly, while the TCBsdid not achieve T cell activation on low CEA-expressing HT-29 cellsalone, addition of CEA-CD28 strongly enhanced the activity of the TCB.We conclude that CEA-CD2 enhances CEA-TCB and CEACAM5-TCB and lowersCEA-expression thresholds on cancer cells for TCBs to activate T cells.

Example 9

In Vitro Functional Characterization of Affinity-Reduced Variants ofCD28(SA)

For the original CD28(SA) binder (TGN1412) an affinity of K_(D)=1 nM wasdetermined. High affinity binders like this harbor the risk to besubject to peripheral sink effects, especially if the target is highlyexpressed in peripheral blood, as is the case for CD28. In order to (i)reduce peripheral sink effects, and (ii) reduce the risk of peripheral Tcell activation through off-tumor binding of bispecific tumor-targedCD28 antibodies to T cells, we generated a series of 31 CD28 binderswith reduced affinities by introducing point mutations in the CDRs (seeExample 3). FIGS. 4A, 4B and 4C show binding to CD28 on CHO cells of theCD28 monospecific, monovalent IgGs from supernatants, confirming thataddition of point mutations generated a broad range of binders withvarying binding properties. Based on these data, a shortlist of 11candidates was selected for conversion into the FAP-CD28 bispecificformat (see Example 4, Table 6) for further characterization. Bindingassays confirmed positive binding to FAP on cells (FIGS. 4F and 4G) aswell as positive and varying binding to CD28 of the chosen variants(FIGS. 4D and 4E).

Affinity-Reduced CD28 Binder Variants are Functional In Vitro in aFAP-CD28 Bispecific Format

To assess whether affinity-reduced CD28 binder variants were stillfunctional and able to support TCB-mediated effector functions, weassessed T cell proliferation in TCB combination. To this end, PBMC Tcells were co-cultured with MCSP- and FAP co-expressing MV3 cells for 5days in the presence of increasing concentrations of FAP-CD28 and fixed,limiting concentration of MCSP-TCB (5 pM). As depicted in FIG. 4H, allvariants of the CD28 binders were functional and able to increaseTCB-mediated T cell proliferation in a concentration dependent manner.Of note, the lowest affinity variant 8 (P1AE3131) shows an approximatereduction of affinity compared to the parental CD28 clone but recoversapproximately 86% of its potency (FIG. 4I). In line with these findings,all variants could further enhance the TCB-mediated killing of MV3target cells (FIG. 4J). The corresponding EC₅₀ values are shown in Table8 below.

TABLE 8 TCB-mediated killing of MV3 target cells by FAP- CD28 bispecificmolecules of MV3 target cells FAP-CD28 EC₅₀ killing variant TAPIR (nM)parental P1AD4492 0.98 variant 8 P1AE3131 2.33 variant 11 P1AE3132 1.73variant 12 P1AE3133 0.98 variant 15 P1AE3134 1.79 variant 29 P1AE31411.11

Based on these results, variants 8 (lowest affinity, 23 nM), 15(intermediate affinity: 7.1 nM) and 29 (removed hot-spots, nearly noaffinity reduction, K_(D)=1.1 nM) were chosen for furthercharacterization in vitro and testing in vivo, judged by efficacy andimproved bio-distribution to the tumor.

Affinity Reduced CD28 Binder Variants are Functional In Vitro in aCEA-CD28 Bispecific Format

In an alternative approach, we converted the three selected variants 8,15 and 29 into a CEA-targeted bispecific format and assessed theirability to enhance CEACAM5-TCB mediated T cell activation and targetcell killing. The binding to CD28 of these molecules is shown in FIG. 16. To assess functionality, PBMC T cells were incubated with increasingconcentrations of CEACAM5-TCB and fixed concentrations of CEA-CD28variants in presence of CEA-expressing MKN45 target cells. As depictedin FIGS. 17A, 17B and 17C, all variants were able to enhanceTCB-mediated CD8 T cell proliferation after 5 days (FIG. 17A), CD4 Tcell proliferation after 5 days (FIG. 17B), and target cell killing at90 h (FIG. 17C). The corresponding EC₅₀ values are summarized in Table 9below.

TABLE 9 EC₅₀ values of TCB-mediated CD8 and CD4 T cell proliferation andtarget cell killing by CEA-CD28 bispecific variants CEA-CD28 CD4proliferation CD8 proliferation Killing antibody EC₅₀ [nM] EC₅₀ [nM]EC₅₀ [nM] Parental CD28 0.0013 0.0015 0.002 Variant 8 0.0035 0.00330.004 Variant 15 0.0021 0.0016 0.005 Variant 29 0.0013 0.0013 0.004 TCBalone 0.046 0.038 0.012

Example 10

Generation and Production of New Anti-CEA Antibodies

10.1 Generation of Humanized Variants of Anti-CEA Antibody A5B7 10.1.1Methodology

Anti-CEA antibody A5B7 is for example disclosed by M. J. Banfield et al,Proteins 1997, 29(2), 161-171 and its structure can be found as PDBID:1CLO in the Protein structural database PDB (world wide web.rcsb.org,H. M. Berman et al, The Protein Data Bank, Nucleic Acids Research, 2000,28, 235-242). This entry includes the heavy and the light chain variabledomain sequence. For the identification of a suitable human acceptorframework during the humanization of the anti-CEA binder A5B7, aclassical approach was taken by searching for an acceptor framework withhigh sequence homology, grafting of the CDRs on this framework, andevaluating which back-mutations can be envisaged. More explicitly, eachamino acid difference of the identified frameworks to the parentalantibody was judged for impact on the structural integrity of thebinder, and back mutations towards the parental sequence were introducedwhenever appropriate. The structural assessment was based on Fv regionhomology models of both the parental antibody and its humanized versionscreated with an in-house antibody structure homology modeling toolimplemented using the Biovia Discovery Studio Environment, version 4.5.

10.1.2 Choice of Acceptor Framework and Adaptations Thereof

The acceptor framework was chosen as described in Table 10 below:

TABLE 10 Acceptor framework Choice of human Closest murine acceptorV-region V-region germline germline A5B7 VH mu-IGHV7-3-02 IGHV3-23-01 orIGHV3-15-01 A5B7 VL mu-IGKV4-72-01 IGKV3-11-01

Post-CDR3 framework regions were adapted from human J-element germlineIGJH6 for the heavy chain, and a sequence similar to the kappa J-elementIGKJ2, for the light chain.

Based on structural considerations, back mutations from the humanacceptor framework to the amino acid in the parental binder wereintroduced at positions 93 and 94 of the heavy chain.

10.1.3 VH and VL Regions of the Resulting Humanized CEA Antibodies

The resulting VH domains of humanized CEA antibodies can be found inTable 11 below and the resulting VL domains of humanized CEA antibodiesare listed in Table 12 below.

TABLE 11Amino acid sequences of the VH domains of humanized CEA antibodies, basedon human acceptor framework IGHV3-23 or IGHV3-15 Seq ID DescriptionSequence No A5B7 VH EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPGKALEWLGF178 murine donor IGNKANGYTTEYSASVKGRFTISRDKSQSILYLQMNTLRAEDSATYYCTRsequence DRGLRFYFDYWGQGTTLTVSS IGHV3-23-02EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA 218 humanISGSGGSTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK acceptor sequenceHumanized variants 3-23A5-1EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 220IGNKANGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRGLRFYFDYWGQGTTVTVSS3-23A5-2 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 221IGNKANGYTTYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRGLRFYFDYWGQGTTVTVSS3-23A5-3 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 222IGNKGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDR GLRFYFDYWGQGTTVTVSS3-23A5-4 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWVGF 223IGNKANGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRGLRFYFDYWGQGTTVTVSS3-23A5-1A EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGF 224(all backmut IGNKANGYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTR ations)DRGLRFYFDYWGQGTTVTVSS 3-23A5-1CEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 225 (A93T)IGNKANGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR DRGLRFYFDYWGQGTTVTVSS3-23A5-1D EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 226 (K73)IGNKANGYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCAR DRGLRFYFDYWGQGTTVTVSS3-23A5-1E EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGF 186 (G54A)IGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTR DRGLRFYFDYWGQGTTVTVSSIGHV3-15*01 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR 219 humanIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT acceptor sequenceHumanized variants 3-15A5-1EVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 227IGNKANGYTTEYSASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTR DRGLRFYFDYWGQGTTVTVSS3-15A5-2 EVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 228IGNKANGYTTEYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTR DRGLRFYFDYWGQGTTVTVSS3-15A5-3 EVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWVGF 229IGNKANGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTR DRGLRFYFDYWGQGTTVTVSS

For the heavy chain, the initial variant 3-23A5-1 was found suitable inbinding assays (but showed slightly less binding than the parentalmurine antibody) and was chosen as starting point for furthermodifications. The variants based on IGHV3-15 showed less bindingactivity compared to humanized variant 3-23A5-1.

In order to restore the full binding activity of the parental chimericantibody, variants 3-23A5-1A, 3-23A5-1C and 3-23A5-1D were created. Itwas also tested for variant 3-23A5-1 whether the length of CDR-H2 couldbe adapted to the human acceptor sequence, but this construct completelylost binding activity. Since a putative deamidation hotspot was presentin CDR-H2 (Asn53-Gly54), we changed that motif to Asn53-Ala54. Anotherpossible hotspot Asn73-Ser74 was backmutated to Lys73-Ser74. Thus,variant 3-23A5-1E was created.

TABLE 12Amino acid sequences of the VL domains of humanized CEA antibodies, basedon human acceptor framework IGKV3-11. Seq ID Description Sequence NoA5B7 VL QTVLSQSPAILSASPGEKVTMTCRASSSVTYIHWYQQKPGSSPKSWIYAT 179 murineSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQHWSSKPPTFGGG donor TKLEIK sequenceIGKV3-11 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD 230 humanASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP acceptor sequencehumanized variants A5-L1EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYAT 231SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQG TKLEIK A5-L2EIVLTQSPATLSLSPGERATLSCRASQSVSSYIHWYQQKPGQAPRLLIYA 232TSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQ GTKLEIK A5-L3EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYDA 233SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQG TKLEIK A5-L4EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYAT 234SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSKPPTFGQG TKLEIK A5-L1AQTVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGSSPKSWIYAT 235 (all_backSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQHWSSKPPTFGQG mutations) TKLEIKA5-L1B QTVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRLLIYAT 236 (Q1T2)SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQG TKLEIK A5-L1CEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGSSPKSWIYAT 237 (FR2)SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQG TKLEIK A5-L1DEIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYAT 187 (46,47)SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQG TKLEIK

The light chain was humanized based on the human IGKV3-11 acceptorframework. In the series A5-L1 to A5-L4, it was learned that variantA5-L1 shows good binding activity (but slightly less than the parentalantibody). Partial humanization of CDR-L1 (variant A5-L2; Kabatpositions 30 and 31) fully abrogates the binding. Likewise, humanizationof CDR-H2 (variant A5-L3; Kabat positions 50 to 56) also fully abrogatesthe binding. The position 90 (variant A5-L4) shows significantcontribution to the binding properties. The Histidine at this positionis important for binding. Thus, variant A5-L1 was chosen for furthermodification.

The series A5-L1A to A5-L1D addressed the question which backmutationsare required to restore the full binding potential of the parentalchimeric antibody. Variant A5-L1A showed that backmutations at Kabatpositions 1, 2, the entire framework 2, and Kabat position 71 do not addany further binding activity. Variants A5-L1B, and A5-L1C addressedsubsets of those positions and confirm that they do not alter thebinding properties. Variant A5-L1D with back mutations at Kabatpositions 46 and 47 showed the best binding activity.

10.1.4 Selection of Humanized A5B7 Antibodies

Based on the new humanization variants of VH and VL new CEA antibodieswere expressed as huIgG1 antibodies with an effector silent Fc (P329G;L234, L235A) to abrogate binding to Fcγ receptors according to themethod described in WO 2012/130831 A1 and their binding to CEA expressedon MKN45 cells was tested and compared to the respective parental murineA5B7 antibody.

TABLE 13 VH/VL combinations expressed as huIgG1_LALA_PG antibodiesA5-L1A A5-L1B A5-LIC A5-LID 3-23A5-1A P1AE2164 P1AE2165 P1AE2166P1AE2167 3-23A5-1C — — P1AE2176 P1AE2177 3-23A5-1D P1AE2179 — P1AE2181P1AE2182

MKN45 (DSMZ ACC 409) is a human gastric adenocarcinoma cell lineexpressing CEA. The cells were cultured in advanced RPMI+2% FCS+1%Glutamax. Viability of MKN-45 cells was checked and cells werere-suspended and adjusted to a density of 1 Mio cells/ml. 100 μl of thiscell suspension (containing 0.1 Mio cells) were seeded into a 96 wellround bottom plate. The plate was centrifuged for 4 min at 400 xg andthe supernatant was removed. Then 40 μl of the diluted antibodies orFACS buffer were added to the cells and incubated for 30 min at 4° C.After the incubation the cells were washed twice with 150 μl FACS bufferper well. Then pi of the diluted secondary PE anti-human Fc specificsecondary antibody (109-116-170, Jackson ImmunoResearch) was added tothe cells. The cells were incubated for an additional 30 min at 4° C. Toremove unbound antibody, the cells were washed again twice with 150 μlper well FACS buffer. To fix the cells 100 μl of FACS buffer containing1% PFA were added to the wells. Before measuring the cells werere-suspended in 150 μl FACS buffer. The fluorescence was measured usinga BD flow cytometer.

In FIG. 18 binding curves of the humanized A5B7 variants are shown. Alltested binders were able to bind to MKN45 cells but binding capacity wasslightly reduced compared to the parental A5B7 antibody. The cloneP1AE2167 had the best binding of all tested variants and was selectedfor further development.

Determination of Affinities of Fab Fragments of Humanized Variants ofMurine CEA-Antibody A5B7 to Human CEA Using Surface Plasmon Resonance(BIACORE)

The affinities of Fab fragments of the humanized variants of murine CEAantibody A5B7 to human CEA were assessed by surface plasmon resonanceusing a BIACORE T200 instrument. On a CM5 chip, human CEA (huN(A2-B2)A-avi-His B) was immobilized at a 40 nM concentration bystandard amine coupling on flow cell 2 for 30 s to about 100 RU. The Fabfragments of the humanized variants of murine CEA antibody A5B7 weresubsequently injected as analytes in 3-fold dilutions ranging from500-0.656 nM for a contact time of 120 s, a dissociation time of 250 or1000 s and at a flow rate of 30 μl/min. Regeneration at the level ofhuman CEA (hu N(A2-B2)A-avi-His B) was achieved by 2 pulses of 10 mMglycine/HCl pH2.0 for 60 s. Data were double-referenced against theunimmobilized flow cell 1 and a zero concentration of the analyte. Thesensorgrams of the analytes were fitted to a simple 1:1 Langmuirinteraction model. Affinity constants [K_(D)] for human CEA (A2 domain)are summarized in Table 14 below.

TABLE 14 Affinity constants of Fab fragments representing differenthumanized variants ofmurine CEA antibody A5B7 to human CEA (A2 domain)Affinity to human N(A2-B2)A-avi-His B Tapir ID Name [M] P1AE0289 CEA(A5B7) Fab (parental 5.59E−10 murine antibody) P1AE4135 Fab derived fromP1AE2164 1.70E−09 P1AE4136 Fab derived from P1AE2165 1.25E−09 P1AE4137Fab derived from P1AE2166 1.13E−08 P1AE4138 Fab derived from P1AE21671.47E−09 P1AE4139 Fab derived from P1AE2176 7.58E−09 P1AE4140 Fabderived from P1AE2177 7.62E−09 P1AE4141 Fab derived from P1AE21791.83E−09 P1AE4142 Fab derived from P1AE2181 2.64E−09 P1AE4143 Fabderived from P1AE2182 2.92E−09

The humanized variants of the murine CEA antibody A5B7 are of loweraffinities than the parental murine antibody. The Fab fragment P1AE4138,derived from P1AE2167 (heavy chain with VH variant 3-23A5-1A and Ckappalight chain with VL variant A5-L1D) was chosen as final humanizedvariant. Moreover, a glycine to alanine mutation at Kabat position 54(G54A) was introduced into the VH domain in order to remove adeamidation site, leading to VL variant 3-23A5-1E. The final humanizedantibody (heavy chain with VH variant 3-23A5-1E and Ckappa light chainwith VL variant A5-L1D) has been named A5H1EL1D or huA5B7.

10.2 Generation of A5H1EL1D-Derived Affinity-Matured Anti-CEA Antibodies10.2.1 Preparation, Purification and Characterization of Antigens forPhage Display Campaign

The murine antibody A5B7 and its humanized derivative A5H1EL1D bind tothe A2 domain of CEACAM5 (CEA) with an affinity of about 0.8 and about2.5 nM, respectively. For the generation of affinity-matured variants ofA5H1EL1D by phage display, 3 different recombinant soluble antigens weregenerated. Each protein contained a C-terminal avi tag for site-specificbiotinylation and a his-tag for purification: The first proteinconsisted of the extra-cellular part of CEACAM1 consisting of the 4Ig-like domains N, A1, B, A2 (NABA-avi-His, SEQ ID NO: 238, Table 15).The second protein was a chimeric protein consisting of 2 CEACAM5 and 2CEACAM1 Ig domains. Based on the sequence of the four domains ofCEACAM1, the DNA encoding the second and third domain of CEACAM1 (A1 andB domains) was replaced by the DNA encoding the A2 and B2 domains ofCEACAM5 (N(A2B2)A-avi-His, SEQ ID NO:239, Table 15). The third proteinwas a chimeric protein consisting of 1 CEACAM5 and 3 CEACAM1 Ig domains.Based on the sequence of the four domains of CEACAM1, the DNA encodingthe third domain of CEACAM1 (B domain) was replaced by the DNA encodingthe B2 domain of CEACAM5 (NA(B2)A-avi-His, SEQ ID NO:240, Table 15). Aschematic description of the three constructs is shown in FIGS. 19A, 19Band 19C.

TABLE 15 Amino acid sequences of used CEA antigens SEQ ID AntigenSequence NO NABA-avi-QLTTESMPFNVAEGKEVLLLVHNLPQQLFGYSWYKGERVDGNRQIVGYAIGT 238 HisQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAMAFTCEPETQDTTYLWWINNQSLPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPSDTYYRPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCHANNSVTGCNRTTVKTIIVTELSPVVAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERMKLSQGNITLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENLINVDLEVLFQGPGSGLNDIFEAQKIE WHEARAHHHHHHN(A2B2)A- QLTTESMPFNVAEGKEVLLLVHNLPQQLFGYSWYKGERVDGNRQIVGYAIGT 239avi-His QQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSALSPVVAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERMKLSQGNITLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENLINVDGSGLNDIFEAQKIEWHEARAHH HHHH NA(B2)A-QLTTESMPFNVAEGKEVLLLVHNLPQQLFGYSWYKGERVDGNRQIVGYAIGT 240 avi-HisQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAMAFTCEPETQDTTYLWWINNQSLPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSALSPVVAKPQIKASKTTVTGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERMKLSQGNITLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENLINVDGSGLNDIFEAQKIEWHEARAHH HHHH

The respective plasmids were transiently transfected into HEK 293 cells,stably expressing the EBV-derived protein EBNA (HEK EBNA). Asimultaneously co-transfected plasmid encoding the biotin ligase BirAallowed avi-tag-specific biotinlylation in vivo. Proteins were purifiedfrom filtered cell culture supernatants referring to standard protocolsusing immobilized metal affinity chromatography (IMAC) followed by gelfiltration. Monomeric protein fractions were pooled, concentrated (ifrequired), frozen and stored at −80° C. Part of the samples wereprovided for subsequent protein analytics and analyticalcharacterization e.g. by SDS-PAGE, size exclusion chromatography (SEC)or mass spectrometry.

10.2.2 Selection of Affinity Matured A5H1EL1D-Derived Antibodies

Humanization of antibody A5B7 resulted in an about 3 to 4-fold reductionof the affinity to CEA measured by SPR. While the affinity for A5B7 wasabout 0.8 nM, an affinity of about 2.5 nM was measured for A5H1EL1D.FACS experiments using cell lines with different CEA expression levelsconfirmed this finding. In order to improve the affinity of thehumanized clone A5H1EL1D, 3 different affinity-maturation libraries weremade and used for the selection of clones with improved affinities byphage display.

10.2.2.1 Generation of A5H1EL1D Affinity Maturation Libraries

Generation of affinity-matured A5H1EL1D-derived antibodies was carriedout by phage display using standard protocols (Silacci et al, 2005). Ina first step, DNA sequences encoding the VH and VL of the humanizedparental clone A5H1EL1D (amino acid sequences SEQ ID Nos: 186 and 187)were cloned into a phagemid which was then used as a template forrandomization. In a next step, three libraries were generated for theselection of favourable clones by phage display. Maturation libraries 1and 2 were randomized either in CDR1 and CDR2 of the heavy chain or inCDR1 and CDR2 of the light chain. The third maturation library wasrandomized in the CDR3 regions of both the heavy and the light chain.The randomized positions in the respective CDR regions are shown inFIGS. 20A and 20B. For the generation of the maturation library 1,randomized in CDR1 and 2 of the heavy chain, two fragments wereassembled by “splicing by overlapping extension” (SOE) PCR and clonedinto the phage vector (FIG. 21A). The following primer combinations wereused to generate the library fragments: fragment 1 (LMB3 (SEQ ID NO:243, Table 16) and A5H1EL1D H1 rev_TN (SEQ ID NO: 241, Table 16) andfragment 2 (A5H1EL1D H2 for_TN (SEQ ID NO: 242, Table 7) andHCDR3-rev-constant (SEQ ID NO: 244, Table 16).

TABLE 16 Primers for A5H1ELID affinity maturation library H1 / H2 SEQ IDName Sequence NO: A5H1EL1D_ CAG CCA CTC GAG GCC TTT ACC CGG TGC TTG GCG241 H1_rev_TN TAC CCA X17 CAT X16 X15 X14 X13 GAA X12 GAAGCC AGA AGC CGC GCA GCT GAG ACG X12: 60% T; 5% A/S/G/Y/N/D/E/QX13: 50% T; 20% S; 4.3% A/G/Y/N/D/E/QX14: 50% D; 20% S; 4.3% G/Y/T/N/A/E/Q X15: 60% Y; 4% G/V/H/S/E/Q/N/D/R/FX16: 50% Y; 20% A; 3.75% G/V/T/H/L/I/R/F X17: 50% N; A5H1EL1D_CGC CAA GCA CCG GGT AAA GGC CTC GAG TGG CTG 242 H2_for_TNGGT X18 ATC X19 X20 X21 X22 X23 GCG TAC ACC ACG GAA TAC TCC GCC TCCX18: 60% F; 10% A; 6 % Y/V/L/I/GX19: 50% G; 20% S; 3% A/K/T/V/N/D/E/Q/L/IX20: 50% N; 20% G; 3.75 % D/E/Q/S/Y/T/H/A X21: 60% K; 5% A/T/Y/N/D/E/Q/RX22: 60% A; 4% V/G/D/P/H/N/E/Q/L/IX23: 60% N; 5% D/E/Q; 4.17% G/T/H/S/A/R LMB3 longCAG GAA ACA GCT ATG ACC ATG ATT AC 243 HCDR3-rev-AAC GGT CAC CGT GGT ACC CTG GCC CCA GTA GTC 244 constantGAA ATA GAA GCG CAG ACC AC

For the generation of the maturation library 2, randomized in CDR1 and 2of the light chain, two fragments were assembled by “splicing byoverlapping extension” (SOE) PCR and cloned into the phage vector (FIG.21B). The following primer combinations were used to generate thelibrary fragments: fragment 1 (LMB3 (SEQ ID NO: 243, Table 17) andA5H1EL1D_L1_rev_TN (SEQ ID NO: 245, Table 17) and fragment 2(A5H1EL1D_L2_for_TN (SEQ ID NO: 246, Table 17) and HCDR3-rev-constant(SEQ ID NO: 244, Table 17).

TABLE 17 Primers for A5H1ELID affinity maturation library L1 / L2 SEQ IDName Sequence NO: A5H1EL1D_ GGA ACG CGG GGC CTG GCC TGG TTT TTG CTG ATA245 1_rev_TN CCA X06 X05 X04 X03 X02 X01 GCT GGA TGC GCGGCA AGA CAG GGT AGC ACG X01: 50% S; 20% V; 3.33% T/A/G/N/D/E/Q/Y/HX02: 50% V; 20% S; 3.33% T/A/G/N/Q/F/Y/P/H X03: 50% T; 20% S; 2.72%A/G/Y/V/P/H/N/D/E/Q/R X04: 60% Y; 4% F/G/A/V/T/H/S/N/Q/RX05: 70% I; 30% L X06: 50% H; 20% A; 3.33% R/K/G/S/T/Q/Y/N/V A5H1EL1D_CAG CAA AAA CCA GGC CAG GCC CCG CGT TCC TGG 246 L2_for_TNATC X07 X08 X09 X10 X11 CTC GCT TCT GGT ATC CCG GCA CGT TTC TCC GGCX07: 60% Y; 10% F; 7.5% H/K/N/SX08: 50% A; 20% D; 3.33% V/G/S/T/Y/H/N/E/QX09: 50% T; 20% A; 3.33% S/G/V/P/H/N/D/E/QX10: 60% S; 4% T/A/G/N/D/E/Q/Y/V/H X11: 60% N; 4% D/E/Q/Y/K/T/H/S/A/RLMB3 long CAG GAA ACA GCT ATG ACC ATG ATT AC 243 HCDR3-rev-AAC GGT CAC CGT GGT ACC CTG GCC CCA GTA GTC 244 constantGAA ATA GAA GCG CAG ACC AC

For the generation of the maturation library 3, randomized in CDR3 ofthe light and heavy chains, two fragments were assembled by “splicing byoverlapping extension” (SOE) PCR and cloned into the phage vector (FIG.21C). The following primer combinations were used to generate thelibrary fragments: fragment 1 (LMB3 (SEQ ID NO:243, Table 18) andLCDR3-rev-constant (SEQ ID NO:249, Table 18) and fragment 2 (A5H1EL1D L3for_TN (SEQ ID NO: 247, Table 18) and A5H1EL1D H3 rev_TN (SEQ ID NO:248, Table 18).

TABLE 18 Primers for ASHIELD affinity maturation library L3 / H3 SEQ IDName Sequence NO: A5H1EL1D_ GAG CCT GAA GAT TTT GCC GTA TAC TAT TGT X24247 L3_for_TN X25 X26 X27 X28 X29 X30 X31 ACT TTC GGT CAGGGC ACC AAG CTG GAA ATC X24: 90% Q; 10% H X25: 60% H; 5% R/K/Q/E/Y/F/N/DX26: 65% W; 7% F/Y/V/L/I X27: 58% S; 4% T/A/G/N/D/E/Q; 2% Y/V/P/H/L/I/RX28: 58% S; 4% T/A/G/N/D/E/Q; 2% Y/V/P/H/L/I/R X29: 60% K; 5% R/H; 2.72%A/V/T/P/Y/N/D/E/Q/L/I X30: 70% P; 5% A/S/T/R/S/LX31: 60% P; 5% L/G/R/M; 2.86% A/V/L/I/F/S/R A5H1EL1D_AAC GGT CAC CGT GGT ACC CTG GCC CCA GTA GTC 248 H3_rev_TNX40 X39 X38 X37 X36 X35 X34 X33 X32 AGT ACAGTA GTA GGT GGC GGT GTC TTC TGC X32: 60% R; 10% K; 2.72%A/V/T/P/Y/N/D/E/Q/L/H X33: 60% D; 5% N/E/Q; 2.5% G/Y/V/T/H/S/A/L/I/RX34: 60% R; 5% K/H; 2.72% A/V/T/P/Y/N/D/E/Q/L/IX35: 60% G; 5% A/S/T; 2.5% Y/V/P/H/N/D/E/Q/L/IX36: 60% L; 4% I/V/A/F; 2.4% G/Y/T/P/H/S/N/D/E/QX37: 60% R; 5% K/H; 2.72% A/V/T/P/Y/N/D/E/Q/L/IX38: 65% F; 5% Y/W/A/V/L/I/G X39: 60% Y; 5% F/W; 2.14%G/A/V/T/P/H/S/N/D/E/Q/L/I/R X40: 80% F; 10% I/L LCDR3-rev-ACA ATA GTA TAC GGC AAA ATC TTC AGG CTC 249 constant LMB3 longCAG GAA ACA GCT ATG ACC ATG ATT AC 243 HCDR3AGA AAC GGT CAC CGT GGT ACC CTG GCC 250 amplification CCA GTA GTC

For the assembly of the fragments of each library, equimolar amounts ofeach fragment were used and amplified with the respective outer primers.For the assembly of the fragments of the third library, randomized inHCDR3 and LCDR3, primer LMB3 (SEQ ID NO:243, Table 18) was used incombination with the primer “HCDR3 amplification” (SEQ ID NO:250, Table18).

This primer was used in order to extend the C-terminal end of VH withthe sequence containing a KpnI site. After assembly of sufficientamounts of full length randomized fragments for all libraries, they weredigested with NcoI/KpnI alongside with identically treated acceptorphagemid vector. A 3-fold molar excess of library insert was ligatedwith 20 μg of phagemid vector. Purified ligations were used for 20transformations resulting in about 0.7×10⁹ to 2×10⁹ transformants.Phagemid particles displaying the A5H1EL1D affinity maturation librarieswere rescued and purified by PEG/NaCl purification to be used forselections.

10.2.2.2 Selection of Affinity Matured A5H1EL1D-Derived Clones

For the selection of affinity-matured clones, phage display selectionwith all 3 libraries was performed using recombinant soluble antigens.Panning rounds were performed in solution according to the followingpattern: 1. Pre-clearing of non-specific phagemid particles byincubation with 200 nM biotinylated NA(B2)A-avi-His and NABA-avi-his for0.5 h, 2. capture of biotinylated NA(B2)A-avi-His, NABA-avi-his, andbound phagemid particles by addition of 5.4×10⁷ streptavidin-coatedmagnetic beads for 10 min, 3. Isolation of non-bound phagemid particlesfrom supernatant for further selection, 4. binding of phagemid particlesto 20 nM biotinylated N(A2B2)A-avi-His for 0.5 h in a total volume of 1ml, 5. capture of biotinylated N(A2B2)A-avi-His protein and specificallybound phage particles by addition of 5.4×10⁷ streptavidin-coatedmagnetic beads for 10 min, 6. washing of beads using 5×1 ml PBS/Tween20and 5×1 ml PBS, 7. elution of phage particles by addition of 1 ml of 100mM TEA for 10 min and neutralization by adding 500 μl 1M Tris/HCl pH7.4, 8. infection of exponentially growing E. coli TG1 bacteria, 9.infection with helperphage VCSM13, and 10. subsequent PEG/NaClprecipitation of phagemid particles to be used in subsequent selectionrounds. Selections were carried out over 3 rounds using decreasingantigen concentrations (20×10⁻⁹ M, 10×10⁻⁹ M, and 2×10⁻⁹ M). In round 3,streptavidin beads were washed with 20×1 ml PBS/Tween20 and 5×1 ml PBS.

Specific binders were identified by ELISA as follows: 100 μl of either10 nM biotinylated N(A2B2)A-avi-His protein or 40 nM biotinylatedNA(B2)A-avi-His protein per well were coated on neutravidin plates.Fab-containing bacterial supernatants were added and binding Fabs weredetected via their Flag-tags using an anti-Flag/HRP secondary antibody.Clones that were ELISA-positive on recombinant N(A2B2)A-avi-His proteinbut not on NA(B2)A-avi-His protein were further tested by SPR.

10.2.2.3 Identification of affinity-matured A5H1EL1D-derived variants bySPR

In order to further characterize the ELISA-positive clones, the off-ratewas measured by surface plasmon resonance using a Proteon XPR36 machineand the results were compared with the parental humanized cloneA5H1EL1D.

For this experiment, about 2000, 1000, and 500 RU of biotinylatedN(A2B2)A-avi-His were immobilized on 3 channels using aStreptavidin-coated NLC chip in vertical orientation. As a control fornon-specific binding, 2000 RU of biotinylated NA(B2)A-avi-His proteinwas immobilized on channel 4. For the off-rate analysis of theidentified ELISA-positive clones, injection direction was changed tohorizontal orientation. Before injection, each Fab-containing bacterialsupernatant was filtered and 3-fold diluted with PBS. The associationtime was 100 s at 100 μl/minute and dissociation times were either 600or 1200 s. Bacterial supernatant without Fab fragment was used forreferencing. Regeneration was performed with 10 mM glycine pH 1.5 for 35s at 50 μl/min (vertical orientation).

Dissociation rate constants (koff) were calculated using a simpleone-to-one Langmuir binding model in ProteOn Manager v3.1 software bysimultaneously fitting the sensorgrams. Clones expressing Fabs with theslowest dissociation rate constants were identified and shortlisted.Shortlisted clones were re-evaluated in an additional SPR experimentsunder the same conditions. This time, during each injection, 4affinity-matured clones were directly compared in parallel with theparental clone A5H1EL1D. Bacterial supernatant without Fab fragment wasused for referencing. Clones that showed a slower dissociation rate thanA5H1EL1D on N(A2B2)A-avi-His and no binding to NA(B2)A-avi-His wereselected and the variable domains of the corresponding phagemids weresequenced. The measured dissociation rates of the best clones are shownin Table 19 and the sequences of the respective variable domains arelisted in Table 20.

TABLE 19 Kinetic dissociation constants (koff) of selected clonesobtained in screening analysis with bacterial supernatant cloneDissociation constant kd (1/s) A5H1EL1D 3.10E−04 P006.038 7.41E−05P005.097 8.87E−05 P005.103 5.37E−05 P002.139 6.47E−05 P001.177 9.81E−05P005.102 4.24E−05

TABLE 20 Amino acid sequences of the parental clone A5H1ELIDand selected affinity-matured clones SEQ ID Clone Chain NO SequenceA5H1EL1D VL 187 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDE TLTISSLEPEDFAVYYCQHWSSKPPTFGQGTKLEIK VH 186 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSS P006.038 VL 195 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDF TLTISSLEPEDFAVYYCQHWSSVPPTFGQGTKLEIK VH 194 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYWGQGTTVTVSS P005.097 VL 197 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDF TLTISSLEPEDFAVYYCQHWSSQPPTFGQGTKLEIK VH 196 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFSFDYWGQGTTVTVSS P005.103 VL 199 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDF TLTISSLEPEDFAVYYCQHWSSISPTFGQGTKLEIK VH 198 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFYFDYWGQGTTVTVSS P002.139 VL 201 EIVLTQSPATLSLSPGERATLSCHASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDE TLTISSLEPEDFAVYYCQHWSSKPPTFGQGTKLEIK VH 200 EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYAMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSS P001.177 VL 203 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDE TLTISSLEPEDFAVYYCQHWSSKPPTFGQGTKLEIK VH 202 EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWVRQAPGKGLEWLGFISNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSS P005.102 VL 205 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDF TLTISSLEPEDFAVYYCQHWSSKSPTFGQGTKLEIK VH 204 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYWGQGTTVTVSS P005.102- VL 207 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYcombo1 QQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDETLTISSLEPEDFAVYYCQHWSSKSPTFGQGTKLEI K VH 206EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMN WVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIR FQFDYWGQGTTVTVSS P005.102- VL 209EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWY combo2QQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDE TLTISSLEPEDFAVYYCQHWSSKSPTFGQGTKLEIK VH 208 EVQLLESGGGLVQPGGSLRLSCAASGFYFSDYYMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYWGQGTTVTVSS P005.103- VL 211 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYcombo1 QQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDETLTISSLEPEDFAVYYCQHWSSISPTFGQGTKLEI K VH 210EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMN WVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIR FSFDYWGQGTTVTVSS P005.103- VL 213EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWY combo2QQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDE TLTISSLEPEDFAVYYCQHWSSISPTFGQGTKLEIK VH 212 EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFSFDYWGQGTTVTVSS P006.038- VL 215 EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYcombo1 QQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSVPPTFGQGTKLEI K VH 214EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYAMN WVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIR FGFDYWGQGTTVTVSS P006.038- VL 217EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWY combo2QQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDF TLTISSLEPEDFAVYYCQHWSSVPPTFGQGTKLEIK VH 216 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYEMNWVRQAPGKGLEWLGFISNKANAYTTEYSASVKGRF TISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYWGQGTTVTVSS

10.2.2.4 Fab Purification of Affinity-Matured A5H1EL1D Clones

In order to further characterize the affinity-matured clones, therespective Fab fragments were purified for the exact analysis of thekinetic parameters. For each clone, a 500 ml culture was inoculated withbacteria harboring the corresponding phagemid and induced with 1 mM IPTGat an optical density measured at 600 nm (0D600) of 0.9. Afterwards, thecultures were incubated at 25° C. overnight and harvested bycentrifugation. After incubation of the re-suspended pellet for 20 minin 25 ml PPB buffer (30 mM Tris-HCl pH8, 1 mM EDTA, 20% sucrose),bacteria were centrifuged again and the supernatant was harvested. Thisincubation step was repeated once with 25 ml of a 5 mM MgSO4 solution.The supernatants of both incubation steps were pooled, filtered andloaded on an IMAC column (His gravitrap, GE Healthcare). Subsequently,the column was washed with 40 ml washing buffer (500 mM NaCl, 20 mMImidazole, 20 mM NaH₂PO₄ pH 7.4). After the elution (500 mM NaCl, 500 mMImidazole, 20 mM NaH₂PO₄ pH 7.4) the eluate was re-buffered using PD10columns (GE Healthcare). The yield of purified protein was in the rangeof 300 to 500 μg/l.

10.2.2.5 SPR Analysis of Purified Affinity-Matured A5H1EL1D FabFragments

Affinity (KD) of purified Fab fragments was measured by surface plasmonresonance using a Proteon XPR36 machine using the same setup asdescribed before.

About 2000, 1000, 500, and 250 RU of biotinylated N(A2B2)A-avi-His wereimmobilized on 4 channels of a Streptavidin-coated NLC chip in verticalorientation. As a control for non-specific binding, 2000 RU ofbiotinylated NA(B2)A-avi-His protein was immobilized on channel 5. Forthe determination of the affinity (KD) of the purified clones, injectiondirection was changed to horizontal orientation. Two-fold dilutionseries of purified Fab fragments (varying concentration ranges between100 and 3 nM) were injected simultaneously at 100 μl/min along separatechannels 1-5, with association times of 100 s, and dissociation times of1200 s. Buffer (PBST) was injected along the sixth channel to provide an“in-line” blank for referencing. Regeneration was performed with 10 mMglycine pH 1.5 for 35 s at 50 μl/min (vertical orientation).

Association rate constants (kon) and dissociation rate constants (koff)were calculated using a simple one-to-one Langmuir binding model inProteOn Manager v3.1 software by simultaneously fitting the associationand dissociation sensorgrams. The equilibrium dissociation constant (KD)was calculated as the ratio koff/kon. The kinetic and thermodynamic dataare listed in Table 21.

TABLE 21 Determination of kinetic and thermodynamic parameters ofpurified Fab-fragments by SPR Clone ID k on (1/Ms) k off (1/s) K_(D)(nM) A5H1EL1D 1.08E+5 2.48E−4 2.3 P006.038 2.25E+5 5.78E−5 0.25 P005.0970.94E+5 8.54E−5 0.91 P005.103 1.00E+5 4.99E−5 0.5 P002.139 1.05E+56.53E−5 0.63 P001.177 2.67E+5 7.85E−4 0.29 P005.102 1.34E+5 3.92E−4 0.29

10.2.2.6 Combination of CDR Positions of Affinity-Matured Clones

In an attempt to further increase the affinity to CEA, CDR positions ofseveral previously identified affinity-matured binders were combinedwith each other. This includes not only specific positions within a CDRbut also combinations of CDRs from different binders. An alignment ofall clones, phage display-derived and combinatorial clones, is shown inFIGS. 22A and 22B. The CDRs of all heavy chains and light chains arelisted in Table 22 and 23, respectively. The VH and VL domains aresummarized in Table 20.

TABLE 22 CDR sequences of affinity-matured heavy chains SEQ SEQ SEQ IDID ID Clone NO CDR-H1 NO CDR-H2 NO CDR-H3 A5H1EL1D 180 GFTFT 181FIGNKANAYT 182 DRGLR DYYMN TEYSASVKG FYFDY P006.038 251 GFTFT 252FIGNKANAYT 253 DRGIR DYYMN TEYSASVKG FGFDY P005.097 257 GFTFT 258FIGNKANAYT 259 DRGLR DYYMN TEYSASVKG FSFDY P005.103 263 GFTFT 264FIGNKANAYT 265 DRGIR DYYMN TEYSASVKG FYFDY P002.139 269 GFYFT 270VISNKANAYT 271 DRGLR DYAMN TEYSASVKG FYFDY P001.177 275 GFYFT 276FISNKANAY 277 DRGLR DYYMN TTEYSASVKG FYFDY P005.102 281 GFTFT 282FIGNKANAYT 283 DRGIR DYYMN TEYSASVKG FQFDY P005.102- 287 GFYFT 288VISNKANAYT 289 DRGIR combo1 DYYMN TEYSASVKG FQFDY P005.102- 293 GFYFS294 VISNKANAYT 295 DRGIR combo2 DYYMN TEYSASVKG FQFDY P005.103- 299GFTFT 300 FIGNKANAYT 301 DRGIR combo1 DYYMN TEYSASVKG FSFDY P005.103-305 GFYFT 306 VISNKANAYT 307 DRGIR combo2 DYYMN TEYSASVKG FSFDYP006.038- 311 GFYFT 312 VISNKANAYT 313 DRGIR combo1 NDYAM TEYSASVKGFGFDY P006.038- 317 GFTFS 318 FISNKANAYT 319 DRGIR combo2 DYEMNTEYSASVKG FGFDY

TABLE 23 CDR sequences of affinity-matured light chains SEQ SEQ SEQ IDID ID Clone NO CDR-L1 NO CDR-L2 NO CDR-L3 A5H1EL1D 183 RASSSVTYI 184ATSNLAS 185 QHWSSKPPT H P006.038 254 RASSSVTYI 255 ATSNLAS 256 QHWSSVPPTH P005.097 260 RASSSVTYI 261 ATSNLAS 262 QHWSSQPPT H P005.103 266RASSSVTYI 267 ATSNLAS 268 QHWSSISPT H P002.139 272 HASSSVTYI 273 ATSNLAS274 QHWSSKPPT H P001.177 278 RASSSVTYI 279 ATSNLAS 280 QHWSSKPPT HP005.102 284 RASSSVTYI 285 ATSNLAS 286 QHWSSKSPT H P005.102- 290RASSSVTYI 291 ATSNLAS 292 QHWSSKSPT combo1 H P005.102- 296 RASSSVTYI 297ATSNLAS 298 QHWSSKSPT combo2 H P005.103- 302 RASSSVTYI 303 ATSNLAS 304QHWSSISPT combo1 H P005.103- 308 RASSSVTYI 309 ATSNLAS 310 QHWSSISPTcombo2 H P006.038- 314 RASSSVTYI 315 ATSNLAS 316 QHWSSVPPT combo1 HP006.038- 320 RASSSVTYI 321 ATSNLAS 322 QHWSSVPPT combo2 H

10.3 Generation and Characterization of Affinity-MaturedA5H1EL1D-Derived Bispecific Antibodies 10.3.1 Cloning, Production, andPurification of Bispecific Antibodies

The variable domains of all clones were synthesized and cloned intoplasmids coding for a bispecific 1+1 IgG molecule based on theknob-into-hole mutations and the crossmab technology in combination withPG-LALA mutations. The second binding moiety was specific for CD28. Aschematic description of the final molecules is drawn in FIG. 1J. Theresulting molecules are composed of the sequences listed in Table 24:All chain combinations expressing the VL and the VH sequences of theaffinity-matured binders (SEQ ID NOs: 323-348) were combined with thetwo chains specific for CD28 (SEQ ID NOs: 349 and 350).

Resulting constructs were prepared by Evitria using their proprietaryvector system with conventional (non-PCR based) cloning techniques andusing suspension-adapted CHO K1 cells (originally received from ATCC andadapted to serum-free growth in suspension culture at Evitria). For theproduction, Evitria used its proprietary, animal-component free andserum-free media (eviGrow and eviMake2) and its proprietary transfectionreagent (eviFect). Proteins were purified from filtered cell culturesupernatants referring to standard protocols. In brief, Fc containingproteins were purified from cell culture supernatants by affinitychromatography using Protein A. Elution was achieved at pH 3.0 followedby immediate neutralization of the sample. The protein was concentratedand aggregated protein was separated from monomeric protein by sizeexclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH6.0.

TABLE 24 Amino acid sequences of the selected affinity-matured anti-CEA clones in bispecific P326G LALA human IgG1 format SEQ ID Clonechain NO Polypeptide sequence A5H1EL VL- 323EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGL CH1-EWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRA 1D IgG1EDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASTKGPSVFPLAPS Fc-SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS (hole,SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD PG-KTHTCPPCPAPEAAGGPSVELFPPKPKDTLMISRTPEVTCVVVDV LALA)SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 324EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRSWIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHWSSKPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC P006.038 VL- 325EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSVPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 326EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P005.097 VL- 327EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSQPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 328EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRESFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P005.103 VL- 329EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS CH1-WIYATSNLASGIPARESGSGSGTDETLTISSLEPEDFAVYYCQHW IgG1SSISPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 330EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P002.139 VL- 331EIVLTQSPATLSLSPGERATLSCHASSSVTYIHWYQQKPGQAPRS CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSKPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 332EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYAMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P001.177 VL- 333EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSKPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 334EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWVRQAPGKGLEWLGFISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGLRFYFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P005.102 VL- 335EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSKSPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 336EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P005.102 VL- 337EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS -combo 1 CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSKSPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 338EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P005.102 VL- 339EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS -combo2 CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSKSPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 340EVQLLESGGGLVQPGGSLRLSCAASGFYFSDYYMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFQFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P005.103 VL- 341EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS -combo1 CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSISPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 342EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANAYTTEYSASVKGRETISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFSFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P005.103 VL- 343EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS -combo2 CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSISPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 344EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYYMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFSFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P006.038 VL- 345EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS -combo 1 CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSVPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 346EVQLLESGGGLVQPGGSLRLSCAASGFYFTDYAMNWVRQAPGKGLEWLGVISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC P006.038 VL- 347EIVLTQSPATLSLSPGERATLSCRASSSVTYIHWYQQKPGQAPRS -combo2 CH1-WIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHW IgG1SSVPPTFGQGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTAALGC Fc-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP (hole,SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA PG-AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV LALA)DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VH-Ck 348EVQLLESGGGLVQPGGSLRLSCAASGFTESDYEMNWVRQAPGKGLEWLGFISNKANAYTTEYSASVKGRFTISRDKSKNTLYLQMNSLRAEDTATYYCTRDRGIRFGFDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENR GEC CD28 VH- 349QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGL (SA CH1-EWIGSIYPGNVQTNYNEKFKDRATLTVDTSISTAYMELSRLRSDD Variant IgG1TAVYFCTRSHYGLDWNFDVWGQGTTVTVSSASTKGPSVFPLAPSS 15) FcKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLOSS (knob,GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDK PG-THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS LALA)HEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP VL-Ck 350DIQMTQSPSSLSASVGDRVTITCHASQNIYVELNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC

10.3.2 Affinity Determination of Selected Antibodies by SPR

The Affinity (K_(D)) of the parental antibody A5H1EL1D as well as itsaffinity-matured derivatives was measured by SPR using a ProteOn XPR36instrument (Biorad) at 25° C.

About 2000, 1000, 500, and 250 RU of biotinylated N(A2B2)A-avi-His wereimmobilized on 4 channels of a Streptavidin-coated NLC chip in verticalorientation. As a control for non-specific binding, 2000 RU ofbiotinylated NA(B2)A-avi-His protein was immobilized on channel 5. Forthe determination of the affinity (K_(D)) of the purified bispecificconstructs, injection direction was changed to horizontal orientation.Two-fold dilution series of purified bi-specific IgGs (varyingconcentration ranges between 25 and 1.56 nM) were injectedsimultaneously at 100 μl/min along separate channels 1-5, withassociation times of 180 s, and dissociation times of 1200 s. Buffer(PBST) was injected along the sixth channel to provide an “in-line”blank for referencing. Regeneration was performed with 10 mM glycine pH1.5 for 20 s at 50 ul/min (vertical orientation).

Association rate constants (kon) and dissociation rate constants (koff)were calculated using a simple one-to-one Langmuir binding model inProteOn Manager v3.1 software by simultaneously fitting the associationand dissociation sensorgrams. The equilibrium dissociation constant(K_(D)) was calculated as the ratio koff/kon. All kinetic andthermodynamic data are listed in Table 25. Higher affinity (lower K_(D)values) were observed for the affinity-matured clones that wereidentified by the phage display selection. In addition, combinationswith exchanged CDRs and CDR positions were tested. While somecombinations (e.g. clones “P005.103-combo2” and “P005.102-combo1”)resulted in very slow off-rates and consequently very high affinities,the affinity was significantly reduced in 2 combinatorial clones (clones“P006.038-combo1” and “P006.038-combo2”).

TABLE 25 Determination of kinetic and thermodynamic parameters ofpurified bi-specific CEA-CD28 constructs by SPR Clone ID k on (1/Ms) koff (1/s) K_(D) (nM) A5H1EL1D 1.58E+5 3.33E−4 2.11 P006.038 2.67E+55.87E−5 0.22 P005.097 2.69E+5 8.87E−5 0.33 P005.103 2.57E+5 1.12E−4 0.44P002.139 2.49E+5 9.70E−5 0.39 P001.177 2.05E+5 8.83E−5 0.43 P005.1021.60E+5 3.31E−5 0.20 P005.102-combo1 2.07E+5 1.23E−5 0.06P005.102-combo2 2.25E+5 1.85E−5 0.08 P005.103-combo1 1.26E+5 3.42E−50.27 P005.103-combo2 1.23E+5 1.05E−5 0.09 P006.038-combo1 1.78E+57.99E−5 4.48 P006.038-combo2 1.91E+5 6.98E−4 3.66

Example 11

Generation and Production of Further Bispecific Antigen BindingMolecules Targeting CD28 and Carcinoembryonic Antigen (CEA)

11.1 Cloning of the Bispecific Antigen Binding Molecules

For the generation of the expression plasmids, the sequences of therespective variable domains were used and sub-cloned in frame with therespective constant regions which are pre-inserted in the respectiverecipient mammalian expression vector. A schematic description of theresulting molecules is shown in FIG. 23 . In the Fc domain, Pro329Gly,Leu234Ala and Leu235Ala mutations (PG-LALA) have been introduced in theconstant region of the human IgG1 heavy chains to abrogate binding to Fcgamma receptors according to the method described in InternationalPatent Appl. Publ. No. WO 2012/130831. For the generation of bispecificantibodies, Fc fragments contained either the “knob” (S354C/T366Wmutations, numbering according to Kabat EU index) or “hole” mutations(Y349C/T366S/L368A/Y407V mutations according to Kabat EU index) to avoidmispairing of the heavy chains. In order to avoid mispairing of lightchains in the bispecific antigen binding molecules, exchange of VH/VL orCH1/Ckappa domains was introduced in one binding moiety (CrossFabtechnology). In another binding moiety, charges were introduced into theCH1 and Ckappa domains as described in International Patent Appl. Publ.No. WO 2015/150447. The generation and preparation of anti-CEA cloneT84.66 is described in WO 2016/075278 A2.

The following molecules were cloned, a schematic illustration thereof isshown in FIGS. 23A to 23D:

-   -   Molecule 11A: CEA (A5H1EL1D)-CD28 (SA) 1+1 format, bispecific        huIgG1 PG-LALA CrossFab molecule with charged modifications in        the CD28(SA) Fab fragment (knob) and VH/VL exchange in        CEA(A5H1EL1D) Fab fragment (hole) (FIG. 23A) comprising the        amino acid sequences of SEQ ID Nos: 351, 352, 353 and 354        (P1AE4773).    -   Molecule 11B: CEA (A5H1EL1D)-CD28 (SA_Variant 8) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 8) Fab fragment (knob) and        VH/VL exchange in CEA(A5H1EL1D) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 351, 352, 355        and 356 (P1AE4774).    -   Molecule 11C: CEA (A5H1EL1D)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in CEA(A5H1EL1D) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 351, 352, 357        and 358 (P1AE4775).    -   Molecule 11D: CEA (A5H1EL1D)-CD28 (SA_Variant 29) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 29) Fab fragment (knob) and        VH/VL exchange in CEA(A5H1EL1D) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 351, 352, 359        and 354 (P1AE4776).    -   Molecule 11E: CEA (A5H1EL1D)-CD28 (SA) 1+1 format, bispecific        huIgG1 PG-LALA CrossFab molecule with VH/VL exchange in the        CD28(SA) Fab fragment (knob) and charged modifications in the        CEA(A5H1EL1D) Fab fragment (hole) (FIG. 23B) comprising the        amino acid sequences of SEQ ID Nos: 360, 361, 362 and 363        (P1AE4777).    -   Molecule 11F: CEA (A5H1EL1D)-CD28 (SA_Variant 8) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28 (SA_Variant 8) Fab fragment (knob) and charged        modifications in the CEA(A5H1EL1D) Fab fragment (hole) (FIG.        23B) comprising the amino acid sequences of SEQ ID Nos: 360,        361, 364 and 365 (P1AE4780).    -   Molecule 11G: CEA (A5H1EL1D)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28 (SA_Variant 15) Fab fragment (knob) and charged        modifications in the CEA(A5H1EL1D) Fab fragment (hole) (FIG.        23B) comprising the amino acid sequences of SEQ ID Nos: 360,        361, 366 and 367 (P1AE4791).    -   Molecule 11H: CEA (A5H1EL1D)-CD28 (SA_Variant 29) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28 (SA_Variant 29) Fab fragment (knob) and charged        modifications in the CEA(A5H1EL1D) Fab fragment (hole) (FIG.        23B) comprising the amino acid sequences of SEQ ID Nos: 360,        361, 368 and 363 (P1AE4793).    -   Molecule 11I: CEA (T84.66)-CD28 (SA) 1+1 format, bispecific        huIgG1 PG-LALA CrossFab molecule with charged modifications in        the CD28(SA) Fab fragment (knob) and VH/VL exchange in        CEA(T84.66) Fab fragment (hole) (FIG. 23A) comprising the amino        acid sequences of SEQ ID Nos: 369, 370, 353 and 354 (P1AE6488).    -   Molecule 11J: CEA (T84.66)-CD28 (SA_Variant 29) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 29) Fab fragment (knob) and        VH/VL exchange in CEA (T.84.66) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 369, 370, 359        and 354 (P1AE6495).    -   Molecule 11K: CEA (T84.66)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in CEA(T84.66) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 369, 370, 357        and 358 (P1AE6557).    -   Molecule 11L: CEA (T84.66)-CD28 (SA_Variant 8) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 8) Fab fragment (knob) and        VH/VL exchange in CEA(T84.66) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 369, 370, 355        and 356 (P1AE6556).    -   Molecule 11M: CEA (T84.66)-CD28 (SA_Variant 29) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28(SA_Variant 29) Fab fragment (knob) and charged        modifications in the CEA(T84.66) Fab fragment (hole) (FIG. 23B)        comprising the amino acid sequences of SEQ ID Nos: 371, 372, 368        and 363 (P1AE9605).    -   Molecule 11N: CEA (T84.66)-CD28 (SA_Variant 8) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28 (SA_Variant 8) Fab fragment (knob) and charged        modifications in the CEA(T84.66) Fab fragment (hole) (FIG. 23B)        comprising the amino acid sequences of SEQ ID Nos371, 372, 366        and 367 (P1AE9606).    -   Molecule 110: CEA (T84.66)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28 (SA_Variant 15) Fab fragment (knob) and charged        modifications in the CEA(T84.66) Fab fragment (hole) (FIG. 23B)        comprising the amino acid sequences of SEQ ID Nos: 371, 372, 364        and 365 (P1AE9607).    -   Molecule 11P: CEA (A5H1EL1D)-CD28 (SA-Variant 29) 2+1,        bispecific monovalent anti-CD28 (SA_Variant 29) and bivalent        anti-CEA huIgG1 PG-LALA CrossFab construct, charged        modifications in both anti-CEA Fab fragments fused head to tail        to each other (hole), VH/VL exchange in the anti-CD28 CrossFab        fragment (knob) (FIG. 23C). The molecule comprises the amino        acid sequences of SEQ ID NOs: 368, 362, 361 and 373 (P1AE6924).    -   Molecule 11Q: CEA (A5H1EL1D)-CD28 (SA_Variant 29) 2+1,        bispecific monovalent anti-CD28 (SA) and bivalent anti-CEA        huIgG1 PG-LALA CrossFab construct, “classical orientation”,        VH/VL exchange in the anti-CD28 CrossFab fragment, charged        modification in both anti-CEA Fab fragments (FIG. 23D). The        molecule comprises the amino acid sequences of SEQ ID NOs: 368,        374, 361 and 360 (P1AE6925).    -   Molecule 11R: CEA (P002.139)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in CEA(P002.139) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 375, 376, 357        and 358 (P1AE8371).    -   Molecule 11S: CEA (P002.139)-CD28 (SA_Variant 8) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 8) Fab fragment (knob) and        VH/VL exchange in CEA(P002.139) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 375, 376, 355        and 356 (P1AF1115).    -   Molecule 11T: CEA (P002.139)-CD28 (SA_Variant 11) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 11) Fab fragment (knob) and        VH/VL exchange in CEA(P002.139) Fab fragment (hole) (FIG. 23A)        comprising the amino acid sequences of SEQ ID Nos: 375, 376, 355        and 354 (P1AF1116). For comparison the following anti-CD28        antibody variants were made:    -   Molecule 11U: CD28(SA_Variant 8) (PG-LALA), CD28 (SA_Variant 8)        antibody in a huIgG1 PG-LALA isotype (FIG. 1B) comprises the        amino acid sequences of SEQ ID NO:377 and SEQ ID NO:378        (P1AE7035).    -   Molecule 11V: CD28(SA_Variant 11) (PG-LALA), CD28        (SA_Variant 11) antibody in a huIgG1 PG-LALA isotype (FIG. 1B)        comprises the amino acid sequences of SEQ ID NO:379 and SEQ ID        NO:380 (P1AE7036).    -   Molecule 11W: CD28(SA_Variant 15) (PG-LALA), CD28        (SA_Variant 15) antibody in a huIgG1 PG-LALA isotype (FIG. 1B)        comprises the amino acid sequences of SEQ ID NO:381 and SEQ ID        NO:382 (P1AE7037).    -   Molecule 11X: CD28(SA_Variant 27) (PG-LALA), CD28        (SA_Variant 27) antibody in a huIgG1 PG-LALA isotype (FIG. 1B)        comprises the amino acid sequences of SEQ ID NO:383 and SEQ ID        NO:384 (P1AE7038).    -   Molecule 11Y: CD28(SA_Variant 29) (PG-LALA), CD28        (SA_Variant 29) antibody in a huIgG1 PG-LALA isotype (FIG. 1B)        comprises the amino acid sequences of SEQ ID NO:385 and SEQ ID        NO:386 (P1AE7039).

11.2 Production of the Molecules

Expression of the above-mentioned molecules is either driven by achimeric MPSV promoter or a CMV promoter. Polyadenylation is driven by asynthetic polyA signal sequence located at the 3′ end of the CDS. Inaddition, each vector contains an EBV OriP sequence for autosomalreplication.

For the production of the Molecules 11A to 11D and 11I to 11T,HEK293-EBNA cells that grow in suspension were co-transfected with therespective expression vectors using polyethylenimine as a transfectionreagent. Antibodies and bispecific antibodies were generated bytransient transfection of HEK293 EBNA cells. Cells were centrifuged andmedium replaced by pre-warmed CD CHO medium. Expression vectors weremixed in CD CHO medium, PEI was added, the solution vortexed andincubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂atmosphere. After the incubation, Excell medium with supplements wasadded (Mammalian Cell Cultures for Biologics Manufacturing, Editors:Weichang Zhou, Anne Kantardjieff). One day after transfectionsupplements (Feed) were added (Mammalian Cell Cultures for BiologicsManufacturing, Editors: Weichang Zhou, Anne Kantardjieff). Cellsupernatants were harvested after 7 days by centrifugation andsubsequent filtration (0.2 μm filter) and purified by standard methods.

Molecules 11U, 11V, 11W, 11X, and 11Y were produced and purified byEvitria using their proprietary vector system with conventional (non-PCRbased) cloning techniques and using suspension-adapted CHO K1 cells(originally received from ATCC and adapted to serum-free growth insuspension culture at Evitria). For the production, Evitria used itsproprietary, animal-component free and serum-free media (eviGrow andeviMake2) and its proprietary transfection reagent (eviFect).Supernatant was harvested by centrifugation and subsequent filtration(0.2 μm filter) and purified by standard methods. Molecules 11E, 11F,11G and 11H were produced and purified by Proteros according to theirstandard methods and protocols.

11.3 Purification of the Molecules

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, Fc containing proteins were purifiedfrom cell culture supernatants by Protein A-affinity chromatography(equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achievedat pH 3.0 followed by immediate pH neutralization of the sample. Theprotein was concentrated by centrifugation (Millipore Amicon® ULTRA-15(Art.Nr.: UFC903096), and aggregated protein was separated frommonomeric protein by size exclusion chromatography in 20 mM histidine,140 mM sodium chloride, pH 6.0.

11.4 Analytical Data of Bispecific Antigen Binding Molecules TargetingCD28 and Carcinoembryonic Antigen (CEA)

The concentration of purified proteins was determined by measuring theabsorption at 280 nm using the mass extinction coefficient calculated onthe basis of the amino acid sequence according to Pace, et al., ProteinScience, 1995, 4, 2411-1423. Purity and molecular weight of the proteinswere analyzed by CE-SDS in the presence and absence of a reducing agentusing a LabChipGXII (Perkin Elmer). Determination of the aggregatecontent was performed by HPLC chromatography at 25° C. using analyticalsize-exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated inrunning buffer (25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-ArginineMonohydrocloride, pH 6.7 or 200 mM KH₂PO₄, 250 mM KCl pH 6.2respectively). A summary of the purification parameters of all moleculesis given in Table 26.

TABLE 26 Summary of the production and purification of CD28 antigenbinding molecules 11A to 11Y Analytical SEC Yield (HMW/Monomer/LMW)Purity measured Molecule Description [mg/l] [%] by CE-SDS [%] 11A CEA(A5H1EL1D)- 11.4 0/100/0 97.8 CD28 (SA) 1 + 1 11B CEA (A5H1EL1D)- 11.30/100/0 98.2 CD28 (SA_Variant 8) 1 + 1 11C CEA (A5H1EL1D)- 45.1 0/100/097.1 CD28 (SA_Variant 15) 1 + 1 11D CEA (A5H1EL1D)- 58.1 0/100/0 96.6CD28 (SA_Variant 29) 1 + 1 11E CEA (A5H1EL1D)- N/A 0.97/95.2/3.93 98.8CD28 (SA, crossed) 1 + 1 11F CEA (A5H1EL1D)- N/A 0.3/96.83/2.86 96.43CD28 (SA_Variant 8, crossed) 1 + 1 11G CEA (A5H1EL1D)- N/A0.1/98.34/1.56 97.9 CD28 (SA_Variant 15, crossed) 1 + 1 11H CEA(A5H1EL1D)- N/A 1.07/96.03/2.9 95.79 CD28 (SA_Variant 29, crossed) 1 + 111I CEA (T84.66) - CD28 N/A (SA) 1 + 1 11J CEA (T84.66) - CD28 25.23.33/96.14/0.53 97.8 (SA_variant 29) 1 + 1 11K CEA (T84.66) - CD28 35.19/93.06/1.75 96.32 (SA_Variant 15) 1 + 1 11L CEA (T84.66) - CD28 7.85.12/95.5/1.84 95.5 (SA Variant 8) 1 + 1 11M CEA (T84.66)-CD28 N/A(SA_variant 29, crossed) 1 + 1 11N CEA (T84.66)-CD28 N/A (SA_Variant 15,crossed) 1 + 1 11O CEA (T84.66)-CD28 N/A (SA_Variant 8, crossed) 1 + 111P CEA (A5H1ELID, N/A head to tail) - CD28 (SA_variant 29) 2 + 1 11QCEA (A5H1EL1D) - N/A CD28 (SA_variant 29) 2 + 1, classical 11RCEA(P002.139)-CD28 7.27 044/99.56/0 100 (SA_Variant 15) 1 + 1 11SCEA(P002.139)-CD28 12 0.57/97.68/1.75 99.44 (SA_Variant 8) 1 + 1 11TCEA(P002.139)-CD28 7.25 0.7/96.61/2.69 95.1 (SA_Variant 11) 1 + 1 11UCD28 (SA_Variant 8) N/A 0/98.43/1.57 80.2 IgG1 PG LALA 11V CD28(SA_Variant 11) N/A 0/98.52/1.48 72.5 IgG1 PG LALA 11W CD28 (SA_Variant15) N/A 1.49/97.44/1.07 82 IgG1 PG LALA 11X CD28 (SA_Variant 27) N/A1.46/97.15/1.39 84.6 IgG1 PG LALA 11Y CD28 (SA_Variant 29) N/A 1.02/97.31.68 84.4 IgG1 PG LALA

11.5 Binding Analysis of Bispecific Antigen Binding Molecules TargetingCD28 and Carcinoembryonic Antigen (CEA) by SPR

Affinity (K_(D)) to CD28 of Molecules 10A-10D, which bear the anti-CEAantibody A5H1EL1D and the anti-CD28 binder variants 8, 11, and 29 aswell as the original binder CD28(SA), were measured by SPR by surfaceplasmon resonance using a Proteon XPR36 machine. For the immobilizationof recombinant antigen (ligand), huCD28-Fc was diluted with PBST (10 mMphosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) toconcentrations ranging from 100 to 500 nM, and then injected at 25μl/minute at varying contact times. This resulted in immobilizationlevels between 500 to 3000 response units (RU) in vertical orientation.

For the determination of the affinity (K_(D)) of the purified molecules,injection direction was changed to horizontal orientation. Two-folddilution series of purified constructs (varying concentration rangesbetween 100 and 6.25 nM) were injected simultaneously at 100 μl/minalong separate channels 1-5, with association times of 150 s, anddissociation times of 400 s. Buffer (PBST) was injected along the sixthchannel to provide an “in-line” blank for referencing. Regeneration wasperformed with 10 mM glycine pH 1.5 for 35 s at 50 μl/min (verticalorientation). Association rate constants (kon) and dissociation rateconstants (koff) were calculated using a simple one-to-one Langmuirbinding model in ProteOn Manager v3.1 software by simultaneously fittingthe association and dissociation sensorgrams. The equilibriumdissociation constant (KD) was calculated as the ratio koff/kon. Thekinetic and thermodynamic data are listed in Table 27.

TABLE 27 kinetic and thermodynamic analysis of anti-CD28 variants inbispecific molecules 11A to 11D k_(on) k_(off) K_(D) Bispecific molecule(1/(s*M) (1/s) (nM) CEA (A5H1EL1D)-CD28 (SA) 1 + 1 3.77E+5 2.94E−4 0.8CEA (A5H1EL1D)-CD28 1.69E+5 9.80E−3 58 (CD28(SA_Variant 8) 1 + 1 CEA(A5H1EL1D)-CD28 2.59E+5 2.64E−3 10 (CD28(SA_Variant 15) 1 + 1 CEA(A5H1EL1D)- CD28 2.71E+5 3.24E−4 1.2 (CD28(SA_Variant 29) 1 + 1

11.6 Biochemical Characterization of the Anti-CD28 IgG Variants andSelected 1+1 Bispecific CEA-Targeted Anti-CD28 Antigen Binding Molecules

In order to characterize and compare their biochemical and biophysicalproperties, the following molecules were analyzed in detail:CEA-targeted anti-CD28 bispecific variants Molecules 10A-10D andanti-CD28 IgG variants Molecules 10U-10Y. The results are summarized inTables 27 and 28.

Hydrophobic Interaction Chromatography (HIC)

Apparent hydrophobicity was determined by injecting 20 μg is of sampleonto a HIC-Ether-(Tosoh) column equilibrated with 25 mM Na-phosphate,1.5 M ammonium sulfate, pH 7.0. Elution was performed with a lineargradient from 0 to 100% buffer B (25 mM Na-phosphate, pH 7.0) within 60minutes. Retention times were compared to protein standards with knownhydrophobicity. Most antibodies display a relative retention timebetween 0 and 0.35.

Thermal Stability

Samples were prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffine oil. The hydrodynamic radius was measuredrepeatedly by dynamic light scattering on a DynaPro Plate Reader (Wyatt)while the samples were heated with a rate of 0.05° C./min from 25° C. to80° C.

FcRn Affinity Chromatography

FcRn was expressed, purified and biotinylated as described by Cymer,Schlothauer et al., Bioanalysis 2017, 9(17),doi.org/10.4155/bio-2017-0109. For coupling, the prepared receptor wasadded to streptavidin-sepharose (GE Healthcare). The resultingFcRn-sepharose matrix was packed in a column housing. The column wasequilibrated with 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES), 140mM NaCl, pH 5.5 (eluent A) at a 0.5 ml/min flow rate. 30 μg is ofantibody samples were diluted at a volume ratio of 1:1 with eluent A andapplied to the FcRn column. The column was washed with 5 column volumesof eluent A followed by elution with a linear gradient from 20 to 100%20 mM Tris/HCl, 140 mM NaCl, pH 8.8 (eluent B) in 35 column volumes. Theanalysis was performed with a column oven at 25° C. The elution profilewas monitored by continuous measurement of the absorbance at 280 nm.Retention times were compared to protein standards with knownaffinities. Most antibodies display a relative retention time between 0and 1.

Heparin Affinity Chromatography

Heparin affinity was determined by injecting 30-50 μg of sample onto aTSKgel Heparin-(Tosoh) column equilibrated with 50 mM Tris, pH 7.4.Elution was performed with a linear gradient from 0 to 100% buffer B (50mM Tris, 1M NaCl, pH 7.4 mM) within 37 minutes. Retention times werecompared to protein standards with known affinities.

All tested sequence variants passed all criteria and do notsignificantly differ from each other with regard to all testedbiophysical and biochemical properties (Tables 28 and 29).

TABLE 28 Biophysical and biochemical properties of tested anti-CD28 IgGvariants 10U-10Y Thermal stability Apparent FcRn Heparin Sample (° C.)hydophobicity affinity affinity CD28 (SA_variant 8) 79 0.218 0.17 0.58IgG1 PG LALA CD28 (SA_variant 11) 78 0.313 0.21 0.58 IgG1 PG LALA CD28(SA_variant 15) 79 0.265 0.25 0.59 IgG1 PG LALA CD28 (SA_variant 27) 780.194 0.28 0.59 IgG1 PG LALA CD28 (SA_variant 29) 78 0.308 0.37 0.59IgG1 PG LALA

TABLE 29 Biophysical and biochemical properties of tested bispecificCEA-targeted anti-CD28 variant molecules 11A-11D Thermal stabilityApparent FcRn Heparin Sample (° C.) hydophobicity affinity affinity CEA(A5H1EL1D) - CD28 70 0.22 0.21 0.66 (SA) 1 + 1 CEA (A5H1EL1D) - CD28 670.17 0.08 0.66 (SA_Variant 8) 1 + 1 CEA (A5H1EL1D) - CD28 70 0.19 0.10.66 (SA_Variant 15) 1 + 1 CEA (A5H1EL1D) - CD28 70 0.22 0.17 0.67(SA_Variant 29) 1 + 1

Example 12

In Vitro Functional Characterization of Bispecific Antigen BindingMolecules Targeting CD28 and Carcinoembryonic Antigen (CEA)

12.1 Binding to CEACAM5 on CEA-Expressing MV3 Cells

To assess whether affinity-maturation of A5H1EL1D resulted in improvedbinding to CEACAM5, a FACS binding assay on MV3 cells, geneticallymodified to express CEA, was performed. As shown in FIG. 24 , a CEA-CD28bispecific antibody (Molecule 11R, P1AE8371) carrying theaffinity-matured anti-CEA clone P002.139 showed superior binding(EC₅₀=4.1 nM) to CEACAM5 than the A5H1EL1D clone (Molecule 11C,P1AE4775) (EC₅₀=20.4 nM).

12.2 IL-2 Reporter Assay to Analyze In Vitro Functionality of CEA-CD28Bispecific Antibodies in Combination with CEA-Targeted TCBs

IL-2 reporter cells (J1631, Promega) are genetically engineered Jurkat Tcells that express a luciferase reporter driven by an IL-2 promoter. Toassess the ability of CEA-targeted CD28 agonists to enhance TCB-mediatedT cell effector function, 10000 MKN45 cells/well were incubated with 10⁵IL-2 reporter cells (E:T ratio 10:1) with fixed concentration of CEA-TCB(5 nM) and a concentration range of CEA-CD28 bispecific antibodies (14pM-10 nM). After 6 h of incubation at 37° C., 5% CO₂, luminescence wasassessed using OneGlo (E6120, Promega) according to manufacturer'sinstructions. Plates were read via Tecan Spark 10M Plate Reader.

The functionality of CEA-CD28 bispecific antibody carrying either theaffinity-matured anti-CEA clone P002.139 (Molecule 11R, P1AE8371) or theclone A5H1EL1D (Molecule 11C, P1AE4775) was assessed using an IL-2reporter cell assay in combination with CEA-TCB (5 nM) and in presenceof CEA-expressing MKN45 cells. As shown in FIGS. 25A and 25B, improvedaffinity of the affinity-matured clone P002.139 translated to higherpotency in costimulatory capacity as compared to the clone A5H1EL1D.

Example 13

In Vivo Functional Characterization of Bispecific Antigen BindingMolecules Targeting CD28 and Carcinoembryonic Antigen (CEA) inCombination with CEA TCB

13.1 Efficacy Study with CEA-CD28 Bispecific Antigen Binding Moleculeswith Different CEA Antigen Binding Domains in Combination with CEA-TCBin MKN45 Xenograft in Humanized Mice

The efficacy study described herein was aimed to understand theCEA-clone dependent potency of the CEA-CD28 bispecific antigen bindingmolecule in combination with CEA-TCB in terms of tumor regression infully humanized NSG mice.

Human MKN45 cells (human gastric carcinoma) were originally obtainedfrom ATCC and after expansion deposited in the Glycart internal cellbank. Cells were cultured in DMEM containing 10% FCS at 37° C. in awater-saturated atmosphere at 5% CO₂. In vitro passage 12 was used forsubcutaneous injection at a viability of 97%. 50 microliters cellsuspension (1×10⁶ MKN45 cells) mixed with 50 microliters Matrigel wereinjected subcutaneously in the flank of anaesthetized mice with a 22G to30G needle.

Female NSG mice, age 4-5 weeks at start of the experiment (JacksonLaboratory) were maintained under specific-pathogen-free condition withdaily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (ZH225-17). After arrival,animals were maintained for one week to get accustomed to the newenvironment and for observation. Continuous health monitoring wascarried out on a regular basis.

Female NSG mice were injected i.p. with 15 mg/kg of Busulfan followedone day later by an i.v. injection of 1×10⁵ human hematopoietic stemcells isolated from cord blood. At week 14-16 after stem cell injectionmice were bled sublingual and blood was analyzed by flow cytometry forsuccessful humanization. Efficiently engrafted mice were randomizedaccording to their human T cell frequencies into the different treatmentgroups. At that time, mice were injected with tumor cells s.c. asdescribed (FIG. 26 , d0) and treated with the compounds or histidinebuffer (Vehicle) when tumor size reached appr. 150 mm³ (day13). All micewere injected i.v. with 200 μl of the appropriate solution. To obtainthe proper amount of compounds per 200 the stock solutions (Table 30)were diluted with histidine buffer when necessary.

TABLE 30 Compositions used in the in vivo experiment ConcentrationCompound Formulation buffer (mg/mL) CEA-TCB 20 mM Histidine, 4.82(=stock 140 mM NaCl, solution) pH 6.0 CEA(T84.66)-CD28 20 mM Histidine,1.71 (=stock (SA_Variant 15) 140 mM NaCl, solution) pH 6.0CEA(A5H1EL1D)-CD28 20 mM Histidine, 3.89 (=stock (SA_Variant 15) 140 mMNaCl, solution) pH 6.0

For combination therapies (Group C and D, FIG. 26 ) with CEA-CD28 andCEA-TCB the bispecific molecules were injected concomitant. Tumor growthwas measured twice weekly using a caliper and tumor volume wascalculated as followed:

Tv:(W ²/2)×L(W: Width, L: Length)

Tumor growth inhibition values as a measurement of potency in vivo werecalculated with a Roche internal statistical program as following:

TGI : ? * 100 ?indicates text missing or illegible when filed

The study was terminated at day 39. FIG. 27A shows the tumor growthkinetics (Mean, +SEM) as well as the individual tumor growth kineticsper group and mouse (FIGS. 27B to 27E). As described here, CEA-TCB, as asingle agent induced little tumor growth inhibition. However, thecombinations with both CEA-CD28 molecules showed improved tumor growthinhibition. Especially the combination of CEA-TCB with CEA-CD28 thatcontains the lower affinity binder for CEA (A5H1EL1D) resulted insuperior tumor growth regression. As displayed in Table 31, highest TGIvalue (TGI: 116%) were calculated for the combination group of CEA-TCBwith CEA-CD28 (huA5B7) indicating strongest anti-tumor effects. TGImeans tumor growth inhibition, TGI>100 means tumor regression andTGI=100 is defined as tumor stasis.

TABLE 31 TGI at study day 39 (Vehicle as Control Group) Group TGI CEATCB 69 CEA TCB + CEA(T84.66)-CD28 89 (SA_Variant 15) CEA TCB +CEA(A5H1EL1D)-CD28 116 (SA_Variant 15)13.2 Efficacy Study with CEA-CD28 Bispecific Antigen Binding Moleculeswith Three Different CD28 Antigen Binding Domains in Combination withCEACAM5-TCB and Anti-PDL1 Antibody in BXPC3 Xenograft in Humanized Mice

This efficacy study was aimed to understand the impact of the affinityof the CD28 antigen binding domain of the CEA-CD28 molecules incombination with CEACAM5-TCB and anti-PD-L1 in terms of tumor regressionand ImmunoPD patterns in fully humanized NSG mice. Three differentaffinity variants have been tested in the current study (Variant8<Variant 15<Variant 29).

Human BXPC3 cells (human pancreatic cancer cell line) were originallyobtained from ECACC (European Collection of Cell Culture) and afterexpansion deposited in the Roche Glycart internal cell bank. BXPC3 cellswere cultured in RPMI containing 10% FCS (PAA Laboratories, Austria)with 1% Glutamax. The cells were cultured at 37° C. in a water-saturatedatmosphere at 5% CO₂. In vitro passage 20 was used for s.c. injection ata viability >95%. 50 microliters cell suspension (1×10⁶ BXPC3 cells)mixed with 50 microliters Matrigel were injected subcutaneously in theflank of anaesthetized mice with a 22G to 30G needle.

Female NSG mice, age 4-5 weeks at start of the experiment (JacksonLaboratory) were maintained under specific-pathogen-free condition withdaily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (ZH225-17). After arrival,animals were maintained for one week to get accustomed to the newenvironment and for observation. Continuous health monitoring wascarried out on a regular basis.

Female NSG mice were injected i.p. with 15 mg/kg of Busulfan followedone day later by an i.v. injection of 1×10⁵ human hematopoietic stemcells isolated from cord blood. At week 14-16 after stem cell injectionmice were bled sublingual and blood was analyzed by flow cytometry forsuccessful humanization. Efficiently engrafted mice were randomizedaccording to their human T cell frequencies into the different treatmentgroups A to F. At that time, mice were injected with tumor cells s.c. asdescribed in FIG. 28 and treated with the compounds or Histidine buffer(Vehicle) when tumor size reached appr. 150 mm³ (day20). All mice wereinjected i.v. with 200 μl of the appropriate solution. To obtain theproper amount of compounds per 200 μl, the stock solutions (Table 32)were diluted with Histidine buffer when necessary.

TABLE 32 Compositions used in the in vivo experiment ConcentrationCompound Formulation buffer (mg/mL) CEACAM5-TCB 20 mM Histidine, 20.5(=stock 140 mM NaCl, solution) pH 6.0 anti-PD-L1 20 mM Histidine, 60(=stock (Atezolizumab) 140 mM NaCl, solution) pH 6.0 CEA(A5H1EL1D)-CD2820 mM Histidine, 3.78 (=stock (SA_Variant 8) 140 mM NaCl, solution) pH6.0 CEA(A5H1EL1D)-CD28 20 mM Histidine, 3.89 (=stock (SA_Variant 15) 140mM NaCl, solution) pH 6.0 CEA(A5H1EL1D)-CD28 20 mM Histidine, 5.01(=stock (SA_Variant 29) 140 mM NaCl, solution) pH 6.0

At termination (day 52), mice were sacrificed, tumors were removed,weighted and single cell suspensions were prepared through an enzymaticdigestion with Collagenase V and DNAse for subsequent FACS-analysis.Single cells where stained for human CD45, CD3, CD8, and CD4 andanalyzed at FACS BDFortessa.

FIG. 29 shows the tumor growth kinetics (Mean, +SEM) for all treatmentgroups, the corresponding TGI values of each treatment arm are shown inTable 33 below. As described here, CEACAM5 TCB monotherapy as well asthe combination with a-PD-L1 induced little tumor growth inhibition.Only the addition of CEA-CD28 (SA_Variant 8) to the combination ofCEACAM5-TCB and a-PD-L1 led to an increased tumor growth inhibition(TGI: 75%). Neither variant 15 nor variant 29 increased the anti-tumoreffects. Interestingly, the Immuno-PD data (FIGS. 30A-D) of tumors fromanimals sacrificed at study termination, revealed that the additionaltumor growth inhibition induced by CEA-CD28 variant 8 is also reflectedby an increased intratumor T cell frequency. FIG. 30A showsrepresentative dot plots of the stained tumor single cell suspensions ofeach treatment arm. The summary of CD3, CD8 and CD4 T cell infiltrationis depicted in FIGS. 30B, 30C and 30D, respectively. No statisticaldifferences were observed in the groups treated with CEA-CD28(SA_Variant 15) or CEA-CD28 (SA_Variant 29) as compared the onlyCEACAM5-TCB or combination with a-PD-L1 in terms of T cell infiltrationin the tumor. The strongest ImmunoPD effects have been detected withCEA-CD28 (SA_Variant 8).

TABLE 33 TGI at study day 52 (Vehicle as Control Group) Group TGICEACAM5 TCB 35 CEACAM5 TCB + a-PD-L1 17 CEACAM5 TCB + a-PD-L1 + 75CEA(A5H1EL1D)-CD28 (SA_Variant 8) CEACAM5 TCB + a-PD-L1 + 37CEA(A5H1EL1D)-CD28 (SA_Variant 15) CEACAM5 TCB + a-PD-L1 + 35CEA(A5H1EL1D)-CD28 (SA_Variant 29)13.3 Efficacy Study with CEA-CD28 Bispecific Antigen Binding Molecule inCombination with CEA TCB in MKN45 Xenograft in Humanized NSG Mice

The efficacy study described herein was aimed to study the combinationof CEA-TCB and CEA-CD28 (SA_Variant 8) in a second human Xenograft modelin terms of tumor regression and ImmunoPD patterns in fully humanizedNSG mice.

Human MKN45 cells (human gastric carcinoma) were originally obtainedfrom ATCC and after expansion deposited in the Glycart internal cellbank. Cells were cultured in DMEM containing 10% FCS at 37° C. in awater-saturated atmosphere at 5% CO₂. In vitro passage 12 was used forsubcutaneous injection at a viability of >95%. 50 microliters cellsuspension (1×10⁶ MKN45 cells) mixed with 50 microliters Matrigel wereinjected subcutaneously in the flank of anaesthetized mice with a 22G to30G needle. Female NSG mice, age 4-5 weeks at start of the experiment(Jackson Laboratory) were maintained under specific-pathogen-freecondition with daily cycles of 12 h light/12 h darkness according tocommitted guidelines (GV-Solas; Felasa; TierschG). The experimentalstudy protocol was reviewed and approved by local government (ZH225-17).After arrival, animals were maintained for one week to get accustomed tothe new environment and for observation. Continuous health monitoringwas carried out on a regular basis.

Female NSG mice were injected i.p. with 15 mg/kg of Busulfan followedone day later by an i.v. injection of 1×10⁵ human hematopoietic stemcells isolated from cord blood. At week 14-16 after stem cell injectionmice were bled sublingual and blood was analyzed by flow cytometry forsuccessful humanization. Efficiently engrafted mice were randomizedaccording to their human T cell frequencies into the different treatmentgroups. At that time, mice were injected with tumor cells s.c. asdescribed in FIG. 31 and treated with the molecules or Histidine buffer(Vehicle) when tumor size reached appr. 150 mm³ (day13). All mice wereinjected i.v. with 200 μl of the appropriate solution. To obtain theproper amount of compounds per 200 the stock solutions (Table 34) werediluted with Histidine buffer when necessary.

TABLE 34 Compositions used in the in vivo experiment ConcentrationCompound Formulation buffer (mg/mL) CEA-TCB 20 mM Histidine, 4.82(=stock 140 mM NaCl, solution) pH 6.0 CEA(A5H1EL1D)-CD28 20 mMHistidine, 3.78 (=stock (SA_Variant 8) 140 mM NaCl, solution) pH 6.0

For the combination therapy (Group C, FIG. 31 ) constructs were injectedconcomitant. Tumor growth was measured twice weekly using a caliper andtumor volume was calculated as followed:

Tv: (W ²/2)×L (W: Width, L: Length)

Tumor growth inhibition (TGI) values as a measurement of potency in vivowere calculated with a Roche internal statistical program as describedin Example 12.1 before.

At termination (day 48), mice were sacrificed, tumors were removed,weighted and single cell suspensions were prepared through an enzymaticdigestion with Collagenase V and DNAse for subsequent FACS-analysis.Single cells were stained for human CD45 and CD3 and analyzed at FACSBDFortessa.

FIG. 32 shows the tumor growth kinetics (Mean, +SEM) for all treatmentgroups, the corresponding TGI values of each treatment arm are shown inTable 35 below. As described here, CEA-TCB monotherapy induced tumorgrowth inhibition with a TGI value of 84%. However, the combinationtreatment with CEA-CD28 (SA_Variant 8) led to a superior tumor growthinhibition (TGI: 101%). Furthermore, the Immuno-PD data (FIG. 3 ) oftumors from animals sacrificed at study termination, revealed that thestrong tumor growth inhibition induced by CEA-CD28 (SA_Variant 8) incombination with CEA-TCB is also reflected by an increased intratumoralT cell frequency. FIG. 33A shows representative dot plots of the stainedtumor single cell suspensions of each treatment arm. The summary of CD3+T cell infiltration is depicted in FIG. 33B.

TABLE 35 TGI at study day 48 (Vehicle as Control Group) Group TGI CEATCB 84 CEA TCB + CEA(A5H1EL1D)-CD28 10 (SA_Variant 8)

Example 14

Generation and Production of Bispecific Antigen Binding MoleculesTargeting CD28 and Epithelial Cell Adhesion Molecule (EpCAM), HER3, CD30or Trophoblast Glycoprotein (TPBG)

14.1 Cloning of Bispecific Antigen Binding Molecules Targeting CD28 andEpCAM, HER3, CD30 or TPBG

For the generation of the expression plasmids, the sequences of therespective variable domains were used and sub-cloned in frame with therespective constant regions which are pre-inserted in the respectiverecipient mammalian expression vector. In the Fc domain, Pro329Gly,Leu234Ala and Leu235Ala mutations (PG-LALA) have been introduced in theconstant region of the human IgG1 heavy chains to abrogate binding to Fcgamma receptors according to the method described in InternationalPatent Appl. Publ. No. WO 2012/130831. For the generation of bispecificantibodies, Fc fragments contained either the “knob” (S354C/T366Wmutations, numbering according to Kabat EU index) or “hole” mutations(Y349C/T366S/L368A/Y407V mutations according to Kabat EU index) to avoidmispairing of the heavy chains. In order to avoid mispairing of lightchains in the bispecific antigen binding molecules, exchange of VH/VL orCH1/Ckappa domains was introduced in one binding moiety (CrossFabtechnology). In another binding moiety, charges were introduced into theCH1 and Ckappa domains as described in International Patent Appl. Publ.No. WO 2015/150447.

The generation and preparation of anti-EpCAM antibody MT201(adecatumumab) is described in U.S. Pat. No. 7,632,925 B2. Theproduction of the anti-HER3 antibody (lumretuzumab) is described in WO2011/076683 A1. Anti-CD30 antibody brentuximab is disclosed in WO02/34661 A2. The generation and preparation of anti-TPBG antibodies,e.g. FAB091, is described in WO 2017/072207 A1.

The following molecules were cloned, a schematic illustration thereof isshown in FIGS. 34A to 34D:

-   -   Molecule 14A: EpCAM (MT201)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28(SA_Variant 15) Fab fragment (knob) and charged        modifications in the EpCAM (MT201) Fab fragment (hole) (FIG.        34A) comprising the amino acid sequences of SEQ ID Nos: 366,        367, 390 and 391 (P1AE9051).    -   Molecule 14B: HER3(lumretuzumab)-CD28 (SA_Variant 15) 1+1        format, bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in the anti-HER3 Fab fragment (hole) (FIG. 34B)        comprising the amino acid sequences of SEQ ID Nos: 357, 358, 392        and 393 (P1AF0151).    -   Molecule 14C: CD30(brentuximab)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in the anti-CD30 Fab fragment (hole) (FIG. 34C)        comprising the amino acid sequences of SEQ ID Nos: 357, 358, 394        and 395 (P1AF1751).    -   Molecule 14D: TPBG(FAB091)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in the anti-TPBG Fab fragment (hole) (FIG. 34D)        comprising the amino acid sequences of SEQ ID Nos: 357, 358, 396        and 397 (P1AF1752).

14.2 Production of the Molecules

Expression of the above-mentioned molecules is either driven by achimeric MPSV promoter or a CMV promoter. Polyadenylation is driven by asynthetic polyA signal sequence located at the 3′ end of the CDS. Inaddition, each vector contains an EBV OriP sequence for autosomalreplication.

Antibodies and bispecific antibodies were generated by transienttransfection of HEK293 EBNA cells or CHO EBNA cells. Cells werecentrifuged and, medium was replaced by pre-warmed CD CHO medium (ThermoFisher, Cat N° 10743029). Expression vectors were mixed in CD CHOmedium, PEI (Polyethylenimine, Polysciences, Inc, Cat N° 23966-1) wasadded, the solution vortexed and incubated for 10 minutes at roomtemperature. Afterwards, cells (2 Mio/ml) were mixed with the vector/PEIsolution, transferred to a flask and incubated for 3 hours at 37° C. ina shaking incubator with a 5% CO₂ atmosphere. After the incubation,Excell medium with supplements (80% of total volume) was added (W. Zhouand A. Kantardjieff, Mammalian Cell Cultures for BiologicsManufacturing, DOI: 10.1007/978-3-642-54050-9; 2014). One day aftertransfection, supplements (Feed, 12% of total volume) were added. Cellsupernatants were harvested after 7 days by centrifugation andsubsequent filtration (0.2 μm filter), and proteins were purified fromthe harvested supernatant by standard methods as indicated below.

14.3 Purification of the Molecules

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, Fc containing proteins were purifiedfrom cell culture supernatants by Protein A-affinity chromatography(equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achievedat pH 3.0 followed by immediate pH neutralization of the sample. Theprotein was concentrated by centrifugation (Millipore Amicon® ULTRA-15(Art.Nr.: UFC903096), and aggregated protein was separated frommonomeric protein by size exclusion chromatography in 20 mM histidine,140 mM sodium chloride, pH 6.0.

14.4 Analytical Data of Bispecific CD28 Antigen Binding Molecules

The concentrations of purified proteins were determined by measuring theabsorption at 280 nm using the mass extinction coefficient calculated onthe basis of the amino acid sequence according to Pace, et al., ProteinScience, 1995, 4, 2411-1423. Purity and molecular weight of the proteinswere analyzed by CE-SDS in the presence and absence of a reducing agentusing a LabChipGXII (Perkin Elmer). Determination of the aggregatecontent was performed by HPLC chromatography at 25° C. using analyticalsize-exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated inrunning buffer (25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-ArginineMonohydrocloride, pH 6.7 or 200 mM KH₂PO₄, 250 mM KCl pH 6.2respectively). A summary of the purification parameters of all moleculesis given in Table 36.

TABLE 36 Summary of the production and purification of CD28 antigenbinding molecules 13A to 13D Analytical Purity SEC (HMW/ measured YieldMonomer/ by CE- Molecule Description [mg/l] LMW) [%] SDS [%] 14A EPCAM(MT201) − 34.6 8.28/91.72/0 94.24 CD28 (SA_Variant 15) 1 + 1 14B HER360.3 0.14/99.65/0.21 98.53 (Lumretuzumab) − CD28 (SA_Variant 15) 1 + 114C CD30 24.4 0/99.45/0.5 94.06 (Brentuximab) − CD28 (SA_Variant 15) 1 +1 14D TPBG (5T4) − 17.5 0/92.6/7.4 98.12 CD28 (SA_Variant 15) 1 + 1

Example 15

In Vitro Functional Characterization of Bispecific Antigen BindingMolecules Targeting CD28 and EpCAM, HER3, CD30 or TPBG

15.1 Binding of EpCAM-CD28 to EpCAM- and CD28-Expressing Cells

The binding of EpCAM-CD28 (Molecule 14A) carrying the intermediateaffinity CD28 Clone Variant 15 (P1AE9051) to EpCAM was tested usingHT-29 cells (ATCC #HTB-38) and the binding to human CD28 was tested withCHO cells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61,modified to stably overexpress human CD28).

To assess binding, cells were harvested, counted, checked for viabilityand re-suspended at 0.5 Mio cells/ml in FACS buffer (eBioscience, Cat No00-4222-26). 5E4 cells were incubated in round-bottom 96-well plates for1 h at 4° C. with increasing concentrations of the EpCAM-CD28 construct(10 pM-500 nM). Then, cells were washed twice with cold FACS buffer,incubated for further 30 min at 4° C. with PE-conjugated, goat-antihuman PE (Jackson ImmunoReserach, Cat No 109-116-098), washed twice withcold FACS buffer, centrifuged and resuspended in 85 ul FACS buffer withDAPI (Roche, Cat No 10236276001) diluted 1:10000. To monitor unspecificbinding interactions between constructs and cells, an anti-DP47 IgG wasincluded as negative control. Binding was assessed by flow cytometrywith a FACS Fortessa (BD, Software FACS Diva). Binding curves wereobtained using GraphPadPrism7.

In vitro cell binding assays verify that the EpCAM-CD28 (P1AE9051)bispecific agonistic antibody binds to human CD28 (FIG. 35A) as well asto human EpCAM on HT-29 cells (FIG. 35B) in a concentration dependentmanner. As expected, no binding was detected with the anti-DP47 IgG,indicating that the detection of binding is due to specific CD28 andEpCAM binding by the respective targeting moieties.

15.2 In Vitro Functional Characterization of EpCAM-CD28 Molecule Basedon IL-2 Reporter Assay

To assess the ability of EpCAM-CD28 (Molecule 14A) to supportanti-CD3-mediated T cell activation, IL-2 reporter cells (Promega, Ca NoJ1651) served as effector cells (Jurkat T cell line that expresses aluciferase reporter driven by the IL-2 promoter) and HT-29 served astumor targets. 1E4 tumor target cells were incubated in whiteflat-bottom 96-well plates for 6 h at 37° C. with 5E4 IL-2 reportercells (E:T 5:1) in presence of 10 nM anti-CD3 (eBioscience #16-0037-85)alone or in combination with increasing concentrations of the EpCAM-CD28construct (24 pM-100 nM). Prior to the measurement, plates wereincubated at room temperature for 15 min, and then 100 μl of substrate(ONE-Glo solution, Promega, Ca No E6120) was added to the cells. After10 min of incubation at room temperature in the dark, luminescence(counts/sec) was measured with a Tecan Spark 10M.

T cell activation in combination with a constant, suboptimal anti-CD3stimulus was assessed. To this end, IL-2 reporter Jurkat cells wereco-cultured with EpCAM-expressing HT29 cells for 6 h in presence ofincreasing concentrations of EpCAM-CD28 (P1AE9051) and fixed, limitingconcentration of anti-CD3 IgG clone OKT3 (10 nM). As depicted in FIG.35C, EpCAM-CD28 was able to enhance T cell activation, as judged byincreased IL-2 production in T cells exposed to suboptimal CD3stimulation in a concentration dependent manner.

15.3 Binding of HER3-CD28 to HER3- and CD28-Expressing Cells

The binding of HER3-CD28 (Molecule 14B) carrying the intermediateaffinity CD28 clone Variant 15 (P1AF0151) to HER3 was tested using T-47Dcells (ATCC #HTB-133) and the binding to human CD28 was tested with CHOcells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61,modified to stably overexpress human CD28).

To assess binding, cells were harvested, counted, checked for viabilityand re-suspended at Mio cells/ml in FACS buffer (eBioscience, Cat No00-4222-26). 5E4 cells were incubated in round-bottom 96-well plates for1 h at 4° C. with increasing concentrations of the HER3-CD28 construct(10 pM-500 nM). Then, cells were washed twice with cold FACS buffer,incubated for further 30 min at 4° C. with PE-conjugated, goat-antihuman PE (Jackson ImmunoReserach, Cat No 109-116-098), washed twice withcold FACS buffer, centrifuged and resuspended in 85 ul FACS buffer withDAPI (Roche, Cat No 10236276001) diluted 1:10000. To monitor unspecificbinding interactions between constructs and cells, an anti-DP47 IgG wasincluded as negative control. Binding was assessed by flow cytometrywith a FACS Fortessa (BD, Software FACS Diva). Binding curves wereobtained using GraphPadPrism7.

FACS Analysis

To assess the relative level of HER3 at the surface of T-47D, 2E5 cellswere centrifuged at 480 xg for 5 min and washed with PBS. Surfacestaining for HER3 (APC anti human, BioLegend #324708) was performedaccording to the supplier's indications. Cells were washed once with 150ul/well of PBS and resuspended in 150 μl/well of PBS and analyzed usingBD FACS Fortessa.

In vitro cell binding assays verify that the HER3-CD28 (Molecule 14B)bispecific agonistic antibody binds to human CD28 (FIG. 36A) as well ashuman HER3 (FIG. 36B) on cells in a concentration dependent manner. Asexpected, no binding was detected with the anti-DP47 IgG, indicatingthat the detection of binding is due to specific CD28 and HER3 bindingby the respective targeting moieties.

15.4 In Vitro Functional Characterization of HER3-CD28 Molecule Based onIL-2 Reporter Assay

To assess the ability of HER3-CD28 (Molecule 14B) to supportanti-CD3-mediated T cell activation, IL-2 reporter cells (Promega, Ca NoJ1651) served as effector cells (Jurkat T cell line that expresses aluciferase reporter driven by the IL-2 promoter) and T-47D served astumor targets. 1E4 tumor target cells were incubated in whiteflat-bottom 96-well plates for 6 h at 37° C. with 5E4 IL-2 reportercells (E:T 5:1) in presence of 10 nM anti-CD3 (eBioscience #16-0037-85)alone or in combination with increasing concentrations of the HER3-CD28construct (24 pM-100 nM). Prior to the measurement, plates wereincubated at room temperature for 15 min, and then 100 ul of substrate(ONE-Glo solution, Promega, Ca No E6120) was added to the cells. After10 min of incubation at room temperature in the dark, luminescence(counts/sec) was measured with a Tecan Spark 10M.

T cell activation in combination with a constant, suboptimal anti-CD3stimulus was assessed. To this end, IL-2 reporter Jurkat cells wereco-cultured with EpCAM-expressing HT29 cells for 6 h in presence ofincreasing concentrations of HER3-CD28 (P1AF0151) and fixed, limitingconcentration of anti-CD3 IgG clone OKT3 (10 nM). As depicted in FIG.36C, EpCAM-CD28 was able to enhance T cell activation, i.e. IL-2production in T cells exposed to suboptimal CD3 stimulation in aconcentration dependent manner.

15.5 In Vitro Functional Characterization of TPBG-CD28 and CD30-CD28Agonistic Antibodies in a PBMC Assay

The ability of TPBG(5T4)-CD28 (Molecule 14D) and CD30-CD28 (Molecule14C) to enhance T cell activation mediated by anti-CD3 stimulation willbe assessed with primary PBMC T cells from healthy donors as effectorcells and 5T4-expressing target cells such as JIMT-1, NCI-H1975,NCI-N87, and Calu-1 cells, or CD30-expressing target cells such asKARPAS-299, respectively. In such an assay, 1E4 tumor target cells willbe incubated in round bottom 96-well plates for 5 days at 37° C. with1E5 (E:T 10:1) in presence of suboptimal concentration of anti-CD3(eBioscience #16-0037-85, clone OKT3) alone or in combination withincreasing concentrations of the CD28 agonistic constructs (24 pM-100nM). Functionality of the CD28-targeted molecules will be assessed byflow cytometry, measuring T cell activation markers (CD25, CD69) and Tcell proliferation (CFSE dilution).

Example 16

Generation and Production of Bispecific Antigen Binding MoleculesTargeting CD28 and a Multiple Myeloma (MM) Cell Surface Antigen

16.1 Cloning of Bispecific Antigen Binding Molecules Targeting CD28 anda Multiple Myeloma (MM) Cell Surface Antigen

For the generation of the expression plasmids, the sequences of therespective variable domains were used and sub-cloned in frame with therespective constant regions which are pre-inserted in the respectiverecipient mammalian expression vector. In the Fc domain, Pro329Gly,Leu234Ala and Leu235Ala mutations (PG-LALA) have been introduced in theconstant region of the human IgG1 heavy chains to abrogate binding to Fcgamma receptors according to the method described in InternationalPatent Appl. Publ. No. WO 2012/130831. For the generation of bispecificantibodies, Fc fragments contained either the “knob” (S354C/T366Wmutations, numbering according to Kabat EU index) or “hole” mutations(Y349C/T366S/L368A/Y407V mutations according to Kabat EU index) to avoidmispairing of the heavy chains. In order to avoid mispairing of lightchains in the bispecific antigen binding molecules, exchange of VH/VL orCH1/Ckappa domains was introduced in one binding moiety (CrossFabtechnology). In another binding moiety, charges were introduced into theCH1 and Ckappa domains as described in International Patent Appl. Publ.No. WO 2015/150447.

Anti-GPRC5D antibody 5E11 is a humanized version of clone 5E11 asdescribed in WO 2019/154890 A1. Anti-CD38 antibodies, e.g. daratumumab,are disclosed in WO 2006/99875 A1. The generation and preparation ofanti-BCMA antibodies is described in WO 2016/166629 A1.

The following molecules were cloned, a schematic illustration thereof isshown in FIGS. 37A to 37C:

-   -   Molecule 16A: GPRC5D (5E11)-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28(SA_Variant 15) Fab fragment (knob) and charged        modifications in the GPRC5D (5E11) Fab fragment (hole) (FIG.        37A) comprising the amino acid sequences of SEQ ID Nos: 366,        367, 398 and 399 (P1AF1272).    -   Molecule 16B: GPRC5D (5E11)-CD28 (SA_Variant 8) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in the GPRC5D (5E11) Fab fragment (hole) (FIG.        37A) comprising the amino acid sequences of SEQ ID Nos: 364,        365, 398 and 399 (P1AF2954).    -   Molecule 16C: CD38 (Daratumumab)-CD28 (SA_Variant 15) 1+1        format, bispecific huIgG1 PG-LALA CrossFab molecule with charged        modifications in the CD28(SA_Variant 15) Fab fragment (knob) and        VH/VL exchange in the anti-CD38 Fab fragment (hole) (FIG. 37B)        comprising the amino acid sequences of SEQ ID Nos: 357, 358, 400        and 401 (P1AE9038).    -   Molecule 16D: anti-BCMA-CD28 (SA_Variant 15) 1+1 format,        bispecific huIgG1 PG-LALA CrossFab molecule with VH/VL exchange        in the CD28(SA_Variant 15) Fab fragment (knob) and charged        modifications in the anti-BCMA Fab fragment (hole) (FIG. 34A)        comprising the amino acid sequences of SEQ ID Nos: 366, 367, 402        and 403 (P1AE9053).

16.2 Production of the Molecules

Expression of the above-mentioned molecules is either driven by achimeric MPSV promoter or a CMV promoter. Polyadenylation is driven by asynthetic polyA signal sequence located at the 3′ end of the CDS. Inaddition, each vector contains an EBV OriP sequence for autosomalreplication.

Antibodies and bispecific antibodies were generated by transienttransfection of HEK293 EBNA cells or CHO EBNA cells. Cells werecentrifuged and, medium was replaced by pre-warmed CD CHO medium (ThermoFisher, Cat N° 10743029). Expression vectors were mixed in CD CHOmedium, PEI (Polyethylenimine, Polysciences, Inc, Cat N° 23966-1) wasadded, the solution vortexed and incubated for 10 minutes at roomtemperature. Afterwards, cells (2 Mio/ml) were mixed with the vector/PEIsolution, transferred to a flask and incubated for 3 hours at 37° C. ina shaking incubator with a 5% CO₂ atmosphere. After the incubation,Excell medium with supplements (80% of total volume) was added (W. Zhouand A. Kantardjieff, Mammalian Cell Cultures for BiologicsManufacturing, DOI: 10.1007/978-3-642-54050-9; 2014). One day aftertransfection, supplements (Feed, 12% of total volume) were added. Cellsupernatants were harvested after 7 days by centrifugation andsubsequent filtration (0.2 μm filter), and proteins were purified fromthe harvested supernatant by standard methods as indicated below.

Alternatively, the antibodies and bispecific antibodies described hereinwere prepared by Evitria using their proprietary vector system withconventional (non-PCR based) cloning techniques and usingsuspension-adapted CHO K1 cells (originally received from ATCC andadapted to serum-free growth in suspension culture at Evitria). For theproduction, Evitria used its proprietary, animal-component free andserum-free media (eviGrow and eviMake2) and its proprietary transfectionreagent (eviFect). Supernatant was harvested by centrifugation andsubsequent filtration (0.2 μm filter) and, proteins were purified fromthe harvested supernatant by standard methods

16.3 Purification of the Molecules

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, Fc containing proteins were purifiedfrom cell culture supernatants by Protein A-affinity chromatography(equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achievedat pH 3.0 followed by immediate pH neutralization of the sample. Theprotein was concentrated by centrifugation (Millipore Amicon® ULTRA-15(Art.Nr.: UFC903096), and aggregated protein was separated frommonomeric protein by size exclusion chromatography in 20 mM histidine,140 mM sodium chloride, pH 6.0.

16.4 Analytical Data of Bispecific CD28 Antigen Binding Molecules

The concentrations of purified proteins were determined by measuring theabsorption at 280 nm using the mass extinction coefficient calculated onthe basis of the amino acid sequence according to Pace, et al., ProteinScience, 1995, 4, 2411-1423. Purity and molecular weight of the proteinswere analyzed by CE-SDS in the presence and absence of a reducing agentusing a LabChipGXII or LabChip GX Touch (Perkin Elmer). Determination ofthe aggregate content was performed by HPLC chromatography at 25° C.using analytical size-exclusion column (TSKgel G3000 SW XL or UP-SW3000)equilibrated in running buffer (25 mM K2HPO₄, 125 mM NaCl, 200 mML-Arginine Monohydrocloride, pH 6.7 or 200 mM KH₂PO₄, 250 mM KCl pH 6.2respectively). A summary of the purification parameters of the moleculesis given in Table 37.

TABLE 37 Summary of the production and purification of CD28 antigenbinding molecules 16A to 16D Analytical Purity SEC (HMW/ measured YieldMonomer/ by CE- Molecule Description [mg/l] LMW) [%] SDS [%] 16A GPRC5D(5E11) − 46.89 0.73/97.68/1.59 93.44 CD28 (SA_Variant 15) 1 + 1 16BGPRC5D (5E11) − 89.84 2.99/95.26/1.75 88.76 CD28 (SA_Variant 8) 1 + 116C CD38 56.55 0.29/99.71/0 93.96 (Daratumumab) − CD28 (SA_Variant 15)1 + 1 16D BCMA − CD28 87.6 1.75/98.25/0 94.33 (SA_Variant 15) 1 + 1

Example 17

In Vitro Functional Characterization of Bispecific Antigen BindingMolecules Targeting CD28 and a Multiple Myeloma (MM) Cell SurfaceAntigen

17.1 Binding of Bispecific Antigen Binding Molecules Targeting CD28 anda Multiple Myeloma (MM) Cell Surface Antigen to Cells, Over-Expressingthe Indicated Target

To measure the binding to GPRC5D, BCMA, CD38 or CD28 we performedFACS-based binding assay on reported multiple myeloma cell lines(Lombardi et al., Molecular characterization of human multiple myelomacell lines by integrative genomics: insights into the biology of thedisease; Genes Chromosomes Cancer. 2007, 46(3), 226-38) or CHOtransfectants, that were transduced to stably overexpress either humanGPRC5D or human CD28: binding to BCMA was assessed, using IM-9 cells(ATCC® CCL-159), binding to CD38 was assessed, using OCI-Ly18 cells(DSMZ ACC 699), binding to GPRC5D was assessed, using CHO-hGPRC5D cellsand binding to CD28 was assessed using CHO-hCD28 cells.

IM-9 and OCI-Ly18 were cultured according to the manufacturers'instructions, the stable CHO transfectants (parental cell line CHO-k1ATCC #CCL-61) were cultured in F-12K, supplemented with 10% FCS.Briefly, suspension cells were harvested, counted and checked forviability. Adherent CHO cells were detached using Cell DissociationBuffer (Gibco), counted and checked for viability. All subsequent stepswere performed at 4° C.

Cells were washed in FACS buffer once (PBS, 2% Fetal Bovine Serum; 1%0.5 m EDTA pH 8; 0.25% NaN₃ Sodium azide) and re-suspended in FACSbuffer at 1 Mio cells per ml. 0.1 Mio cells were plated per well of around-bottom 96-well-plate, washed with FACS buffer once more andsupernatants were discarded. Cells were stained in a total volume of 50ul per well and increasing concentrations of the indicated CD28bispecific molecules (0.07-300 nM) for 30 minutes at 4° C. Cells werewashed twice with FACS buffer and incubated for 30 min at 4° C. in atotal of 25 ul per well, containing the pre-diluted secondary antibody(Alexa Fluor 488-AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, FcγFragment Specific (Jackson Immunoresearch, 109-546-008, diluted 1:100 inFACS buffer, respective 109-606-008 PE, Jackson Immunoresearch, diluted1:100 in FACS buffer, as indicated). Cells were washed twice andanalyzed on a BD Fortessa flow cytometer, equipped with the softwareFACS Diva. Binding curves and EC₅₀ values were obtained usingGraphPadPrism6.

FIGS. 38A to 38F show that all bispecific CD28 molecules are able tobind both, human CD28 (A), as well as the respective second target,namely human CD38, human BCMA or human GPRC5D in aconcentration-dependent manner. Briefly, the GPRC5D-CD28 molecule hasthe lowest EC₅₀ for binding to human CD28 (Table 37), but reaches lowermaximal binding as compared to the other two CD28 bispecific molecules.

Both, CD38- and BCMA-targeted CD28 molecules showconcentration-dependent binding to CD38 and BCMA respectively, and reachsaturation in the evaluated concentration range (EC₅₀ binding values aresummarized in Table 38). In contrast, the GPRC5D-CD28 molecule isbinding in a concentration-dependent manner, but does not reachsaturation in the same concentration range, indicating a lower affinityof the GPRC5D versus the CD38 or BCMA binder, included in thesemolecules.

TABLE 38 EC50 values (nM) for binding of the indicated bispecific CD28molecules to either human CD28 or the respective MM target antigens,expressed on cells EC₅₀ Binding EC₅₀ Binding to to CD28 CD38/BCMA/GPRC5DMolecule (nM) (nM) CD38-CD28 26.8 4.3 BCMA-CD28  9.9 3.96 GPRC5D-CD284.7-5.2 not calculated (SA_variant 15) GPRC5D-CD28 74.2 not calculated(SA_variant 8)

17.2 In Vitro Functional Characterization Based on IL-2 Reporter Assay(Functional Characterization of T-Cell Activation)

To assess the ability of CD38-CD28, BCMA-CD28 and GPRC5D-CD28 bispecificantigen binding molecules to support TCB-mediated T cell activation,IL-2 reporter cells (Promega, Ca No J1651) were used as effector cells(Jurkat T cell line that expresses a luciferase reporter driven by theIL-2 promoter) and NCI-H929 cells, being positive for CD38, BCMA, aswell as GPRC5D as tumor targets.

Briefly, 5×10³ tumor target cells were incubated in white flat-bottom384-well plates (353988 Falcon™ 384-Well Flat-Bottom Tissue CultureTreated Microplate) for 5 h, respective 22 h at 37° C. with 2.5×10⁴ IL-2reporter cells (E:T 5:1) in presence of 1 nM GPRC5D-TCB alone or incombination with increasing concentrations of the CD28 bispecificmolecules (12.2-50 nM). Prior to the measurement, plates were incubatedat room temperature for 15 min, and then 20 ul of substrate (ONE-Glosolution, Promega, Cat No E6120) was added to the cells. After 10 min ofincubation at room temperature in the dark, Luminescence (counts/sec)was measured with a Tecan Spark 10M.

As depicted in FIGS. 39A to 39F, none of the MINI-targeted CD28molecules induces IL2 Jurkat reporter cell activation in the absence ofa TCB signal (bright grey line, FIG. 39A-9F). As expected, with theseIL-2 reporter cells, there is also no significant induction of reportercell activation in the presence of 1 nM GPRC5D TCB, neither at the earlytimepoint (5 h), nor at the later timepoint (after 22 h).

However, all MM-targeted molecules are able to induce significant,concentration- and time-dependent activation of IL-2 reporter cells inpresence of 1 nM GPRC5D-TCB. While CD38-CD28 and BCMA-CD28 molecules areable to induce IL2-reporter cell activation already after 5 h (FIGS. 39Aand 39C), GPRC5D-CD28 reaches similar maximal levels of activation onlyat the later timepoint assessed (22 h, FIG. 39F), indicating differentkinetics of activity.

The anti-GPRC5D/anti-CD3 bispecific antibody (GPRC5D TCB, FIG. 37D) usedin the experiments has been prepared in analogy to CEACAM5 TCB asdescribed in Example 8 and in WO 2016/079076 A1. GPRC5D TCB comprisesthe amino acid sequences of SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:404and SEQ ID NO:405.

17.3 T-Cell Mediated Lysis of Multiple Myeloma Cell Line

To assess the ability of CD38-CD28, BCMA-CD28 and GPRC5D-CD28 to boostGPRC5D TCB-mediated lysis of a Multiple Myeloma Cell line, 1.5×10⁵ humanpan T effector cells were incubated with 3×10⁴ NCI-H929 target cells ata final E:T ratio of 1:1 for roughly 22 h. Pan T cells were isolatedfrom PBMCs by MACS, using the Pan T cell isolation Kit (Miltenyi Biotec,Cat No 130-096-535) according to the manufacturer's instructions. GPRC5DTCB was added at increasing concentrations (0.064 pM-1 nM), thedifferent MM-targeted bispecific CD28 antigen binding molecules wereadded at a fixed concentration of 0.2 nM. Tumor Cell Lysis was assessedas follows: Assay plates were centrifuged for 5 minutes and 50 ulsupernatant per well were transferred into a new 96-flat-bottom-wellplate. For normalization, maximal lysis of the target cells (=100%) wasinduced by incubation of the target cells with a final concentration of1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubatedwith effector cells, but without any bispecific construct or TCB. Afteran overnight incubation of roughly 22 h at 37° C., 5% CO₂, LDH releaseof apoptotic/necrotic target cells into the supernatant was measuredusing the LDH detection kit (Roche Applied Science, #11 644 793 001),according to the manufacturer's instructions.

As illustrated in FIGS. 40 a to 40C, the GPRC5D TCB inducesconcentration-dependent lysis of NCI-H929 cells. All three evaluatedMM-targeted bispecific CD28 antigen binding molecules are able tosignificantly boost efficacy compared to the TCB monotherapy, whenadministered at a fixed concentration of 0.2 nM. Furthermore, none ofthe MM-targeted bispecific CD28 antigen binding molecules is inducingtumor cell lysis in the absence of the TCB, which supports thedependency of the MM-targeted molecules on the TCB signal.

Table 39 summarizes the EC₅₀ values, as well as area under the curvederived from the data shown in FIGS. 40A-40C. EC₅₀ values werecalculated using GraphPadPrism6.

TABLE 39 EC50 values (nM) of anti-GPRC5D TCB mediated killing GPRC5D+CD38- +BCMA- +GPRC5D- TCB CD28 CD28 CD28 EC₅₀ 1.86 3.1 3.7 3.3 lysisafter 22 h (pM) Area under 21.3 44.8 46.3 46.8 the curve

Example 18

Generation and Production of Bispecific Antigen Binding MoleculesTargeting CD28 and CD19 or CD79b

18.1 Cloning of Bispecific Antigen Binding Molecules Targeting CD28 andCD19 or CD79b

The generation and preparation of CD19 antibodies is disclosed in WO2017/55541 A1 or WO 2017/55328 A1. In particular, the CD19 clone 2B11 isdescribed in WO 2017/55328 A1, which is incorporated herein byreference. The CD79b clone huMA79b.v28 (corresponding to polatuzumab) asused herein is described in WO 2009/012268 A. For the generation of therespective expression plasmids, the sequences of the respective variabledomains were used and sub-cloned in frame with the respective constantregions which are pre-inserted in the respective recipient mammalianexpression vector. A schematic description of the resulting molecules isshown in FIGS. 41A and 41B, respectively. Pro329Gly, Leu234Ala andLeu235Ala mutations (PG-LALA) have been introduced in the constantregion of the human IgG1 heavy chains to abrogate binding to Fc gammareceptors. For the generation of unsymmetric bispecific antibodies,Fc-fragments contained either the “knob” or “hole” mutations to avoidmispairing of the heavy chains. In order to avoid mispairing of lightchains in bi- and multispecific antibody constructs, exchange of VH/VLor CH1/Ckappa domains was introduced in one binding moiety (CrossFabtechnology). In another binding moiety, charges were introduced into theCH1 and Ckappa domains.

The following molecules were cloned, a schematic illustration thereof isshown in FIGS. 41A and 41B:

-   -   Molecule 18A: CD19 (8B8-2B11)-CD28 (SA_v29) 1+1, bispecific        huIgG1 PG-LALA CrossFab molecule with charge modifications in        the CD28 v29 Fab (knob) and VH/VL exchange in the CD19 (2B11)        Fab (hole) (FIG. 41A). The molecule comprises the heavy chain        amino acid sequences of SEQ ID NOs: 118 and 430 and the light        chain amino acid sequences of SEQ ID NOs:65 and 431 (P1AE8002).    -   Molecule 18B: CD19 (8B8-2B11)-CD28 (SA_v15) 1+1, bispecific        huIgG1 PG-LALA CrossFab molecule with charge modifications in        the CD28 v15 Fab (knob) and VH/VL exchange in the CD19 (2B11)        Fab (hole) (FIG. 41A). The molecule comprises the heavy chain        amino acid sequences of SEQ ID NOs: 116 and 430 and the light        chain amino acid sequences of SEQ ID NOs:121 and 431 (P1AE9040).    -   Molecule 18C: CD19 (8B8-2B11)-CD28 (SA_v8) 1+1, bispecific        huIgG1 PG-LALA CrossFab molecule with charge modifications in        the CD28 v8 Fab (knob) and VH/VL exchange in the CD19 (2B11) Fab        (hole) (FIG. 41A). The molecule comprises the heavy chain amino        acid sequences of SEQ ID NOs: 114 and 430 and the light chain        amino acid sequences of SEQ ID NOs:122 and 431 (P1AF0175).    -   Molecule D: CD19 (8B8-2B11)-CD28 (SA_v11) 1+1, bispecific huIgG1        PG-LALA CrossFab molecule with charge modifications in the CD28        v11 Fab (knob) and VH/VL exchange in the CD19 (2B11) Fab (hole)        (FIG. 2B). The molecule comprises the heavy chain amino acid        sequences of SEQ ID NOs: 114 and 430 and the light chain amino        acid sequences of SEQ ID NOs:65 and 431 (P1AF0377).    -   Molecule E: CD19 (8B8-2B11)-CD28 (SA_v27) 1+1, bispecific huIgG1        PG-LALA CrossFab molecule with charge modifications in the CD28        v27 Fab (knob) and VH/VL exchange in the CD19 (2B11) Fab (hole)        (FIG. 2B). The molecule comprises the heavy chain amino acid        sequences of SEQ ID NOs: 118 and 430 and the light chain amino        acid sequences of SEQ ID NOs:123 and 431 (P1AF0378).    -   Molecule F: CD79b (huMA79b.v28)-CD28 (SA_v15) 1+1, bispecific        huIgG1 PG-LALA CrossFab molecule with charge modifications in        the CD28 v15 Fab (knob) and VH/VL exchange in the CD79b        (huMA79b.v28) Fab (hole) (FIG. 2C). The molecule comprises the        heavy chain amino acid sequences of SEQ ID NOs: 116 and 432 and        the light chain amino acid sequences of SEQ ID NOs:121 and 433        (P1AE9039).

18.2 Production of Bispecific Antigen Binding Molecules Targeting CD28and CD19 or CD79b

Expression of the above-mentioned molecules is driven by a CMV promoter.Polyadenylation is driven by a synthetic polyA signal sequence locatedat the 3′ end of the CDS. In addition, each vector contains an EBV OriPsequence for autosomal replication.

For the production of all constructs, HEK293-EBNA cells that grow insuspension were transiently co-transfected with the respectiveexpression vectors using polyethylenimine as a transfection reagent.Cells were centrifuged and medium replaced by pre-warmed CD CHO medium.Expression vectors were mixed in CD CHO medium, PEI was added, thesolution vortexed and incubated for 10 minutes at room temperature.Afterwards, cells were mixed with the DNA/PEI solution, transferred toshake flask and incubated for 3 hours at 37° C. in an incubator with a5% CO₂ atmosphere. After the incubation, Excell medium with supplementswas added (Mammalian Cell Cultures for Biologics Manufacturing, Editors:Weichang Zhou, Anne Kantardjieff). One day after transfection,supplements (Feed) were added (Mammalian Cell Cultures for BiologicsManufacturing, Editors: Weichang Zhou, Anne Kantardjieff). Cellsupernatants were harvested after 7 days by centrifugation andsubsequent filtration (0.2 μm filter) and purified by standard methods.

18.3 Purification of Bispecific Antigen Binding Molecules Targeting CD28and CD19 or CD79b

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, Fc containing proteins were purifiedfrom cell culture supernatants by Protein A-affinity chromatography(equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achievedat pH 3.0 followed by immediate pH neutralization of the sample. Theprotein was concentrated by centrifugation (Millipore Amicon® ULTRA-15(Art.Nr.: UFC903096), and aggregated protein was separated frommonomeric protein by size exclusion chromatography in 20 mM histidine,140 mM sodium chloride, pH 6.0.

18.4 Analytical Data of Bispecific or Trispecific Antibodies TargetingCD28 and CD19 or CD79b

The protein concentration of purified constructs was determined bymeasuring the optical density (OD) at 280 nm, using the mass extinctioncoefficient calculated on the basis of the amino acid sequence accordingto Pace, et al., Protein Science, 1995, 4, 2411-1423. Purity andmolecular weight of the proteins were analyzed by CE-SDS in the presenceand absence of a reducing agent using a LabChipGXII (Perkin Elmer).Determination of the aggregate content was performed by HPLCchromatography at 25° C. using analytical size-exclusion column (TSKgelG3000 SW XL or UP-SW3000) equilibrated in running buffer (25 mM K₂HPO₄,125 mM NaCl, 200 mM L-Arginine Monohydrocloride, pH 6.7 or 200 mMKH₂PO₄, 250 mM KCl pH 6.2 respectively). A summary of the purificationparameters of all molecules is given in Table 40.

TABLE 40 Summary of the production and purification of bispecific CD28antigen binding molecules Analytical Purity SEC (HMW/ measured YieldMonomer/ by CE- Molecule Description [mg/l] LMW) [%] SDS [%] 18A CD19(2B11) − 24.9 1.45/98.55/0 96.25 CD28 (v29) 1 + 1 18B CD19 (2B11) −45.05 0/99.1/0.9 98.78 CD28 (v15) 1 + 1 18C CD19 (2B11) − 22.062.08/96.67/1.25 93.11 CD28 (v8) 1 + 1 18D CD19 (2B11) − 40.9 0/98.9/1.197.1 CD28 (v11) 1 + 1 18E CD19 (2B11) − 41.7 0.49/98.9/0.61 90.9 CD28(v27) 1 + 1 18F CD79b 20.6 1.31/98.34/0.35 94.43 (huMA79b.v28) −CD28(v15) 1 + 1

Example 19

Binding and Kinetic Analysis of Bispecific Antigen Binding MoleculesTargeting CD28 and CD19 or CD79b

19.1 Kinetic Analysis of Bispecific Antigen Binding Molecules TargetingCD79b

Affinity (K_(D)) of huMA79b.v28 to recombinant CD79b-His (Sinobiological#29750-H08H) was measured by SPR by surface plasmon resonance using aBiacore T200 machine. For the capture of CD79b-His, an anti-penta HISantibody was coupled to a flow cell of a CM5 chip by amine coupling.Immobilization levels of approx. 5000 units were used. CD79b-His wasthen diluted to a concentration of 1 nM and was captured by theanti-penta HIS antibody during 10 s at a flow rate of 10 μl/min.

For the determination of the affinity (K_(D)) of the purified Molecule F(CD79b (huMA79b.v28)-CD28 (SA_variant 15) 1+1) a two-fold dilutionseries of the purified antigen binding molecule (varying concentrationranges between 125 and 0.49 nM) were injected at 30 μl/min with anassociation time of 180 s, and a dissociation time of 400 s. EIBS-EP⁺buffer (GE-Healthcare standard buffer BR-1006-69 1:10 diluted) wasinjected for referencing. Regeneration was performed with 10 mM glycinepH 2.1 for 2×60 s. Association rate constants (k_(on)) and dissociationrate constants (k_(off)) were calculated using a simple one-to-oneLangmuir binding model by simultaneously fitting the association anddissociation sensorgrams (FIG. 42 ). The equilibrium dissociationconstant (K_(D)) was calculated as the ratio k_(off)/k_(on). The resultsare shown in Table 41. The affinity of Molecule F (CD79b(huMA79b.v28)-CD28 (SA_variant 15) 1+1) to CD79b-His is 76 nM.

TABLE 41 Characterization of the binding of huMA79b.v28 to recombinantsoluble CD79b-His clone k on (1/Ms) k off (1/s) K_(D) (nM) huMA79b.v282.57E+5 1.95E−2 76

19.2 Kinetic Analysis of Bispecific Antigen Binding Molecules TargetingCD28

Affinity (K_(D)) of the produced bispecific antigen binding molecules toCD28 was measured by SPR using a ProteOn XPR36 instrument (Biorad) at25° C. with biotinylated huCD28-Fc antigen immobilized on NLC chips byneutravidin capture. Immobilization of recombinant antigens (ligand):Antigen was diluted with PBST (10 mM phosphate, 150 mM sodium chloridepH 7.4, 0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute atvarying contact times, to achieve immobilization levels of about 200,400 or 800 response units (RU) in vertical orientation. Injection ofanalytes: For one-shot kinetics measurements, injection direction waschanged to horizontal orientation, two-fold dilution series of purifiedbispecific CD19-targeted anti-CD28 affinity variants (varyingconcentration ranges between 50 and 3.125 nM) were injectedsimultaneously at 50 μl/min along separate channels 1-5, withassociation times of 150 s, and dissociation times of 450 s. Buffer(PBST) was injected along the sixth channel to provide an “in-line”blank for referencing. Association rate constants (k_(on)) anddissociation rate constants (k_(off)) were calculated using a simpleone-to-one Langmuir binding model in ProteOn Manager v3.1 software bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) was calculated as the ratiok_(off)/k_(on). Analyzed clones revealed K_(D) values in a broad range(between 1 and 50 nM).

Example 20

Binding of Bispecific CD28 Agonistic Antigen Binding Molecules toCD28-Expressing and CD19- or CD79b-Expressing Cells

Binding to human CD28 was tested with CHO cells expressing human CD28(parental cell line CHO-k1 ATCC #CCL-61, modified to stably overexpresshuman CD28). To assess binding, cells were harvested, counted, checkedfor viability and re-suspended at 2.5×10⁵/ml in FACS buffer(eBioscience, Cat No 00-4222-26). 5×10⁴ cells were incubated inround-bottom 96-well plates for 2 h at 4° C. with increasingconcentrations of the CD28 binders (1 pM-100 nM). Then, cells werewashed three times with cold FACS buffer, incubated for further 60 minat 4° C. with PE-conjugated, goat-anti human PE (Jackson ImmunoReserach,Cat No 109-116-098), washed once with cold FACS buffer, centrifuged andresuspended in 100 ul FACS buffer. To monitor unspecific bindinginteractions between constructs and cells, an anti-DP47 IgG was includedas negative control. Binding was assessed by flow cytometry with a FACSFortessa (BD, Software FACS Diva). Binding curves were obtained usingGraphPadPrism6.

The monovalent one-armed IgG-like CD28 variant constructs showeddifferences in binding as can be seen from FIGS. 4A to 4C.

The binding to CD19 and CD79b was tested using B cell lines expressingdifferent levels of CD19 and CD79b: Nalm6 (DSMZ #ACC 128), RCK8 (DSMZ#ACC 561), WSU DLCL2 (DSMZ #ACC 575) and Z138 (gift from M. Dyer, Univ.of Leicester).

To assess binding, cells were harvested, counted, checked for viabilityand re-suspended at 0.5 Mio cells/ml in FACS buffer (eBioscience, Cat No00-4222-26). 5E4 cells were incubated in round-bottom 96-well plates for1 h at 4° C. with increasing concentrations of the CD19-CD28 (orCD79b-CD28) constructs (10 pM-500 nM). Then, cells were washed twicewith cold FACS buffer, incubated for further 30 min at 4° C. withPE-conjugated, goat-anti human PE (Jackson ImmunoReserach, Cat No109-116-098), washed twice with cold FACS buffer, centrifuged andresuspended in 85 ul FACS buffer with DAPI (Roche, Cat No 10236276001)diluted 1:10000. To monitor unspecific binding interactions betweenconstructs and cells, an anti-DP47 IgG was included as negative control.Binding was assessed by flow cytometry with a FACS Fortessa (BD,Software FACS Diva). Binding curves were obtained using GraphPadPrism7.A comparison of the binding of CD19-CD28 v15 to the different B celllines is shown in FIGS. 43A and 43B.

FACS Analysis

To assess the relative level of CD19 at the surface of B cell lines(Nalm6, RCK8, WSU DLCL2 and Z138), cells were Fc-blocked prior to thestaining using Human Fc Block (BD, Cat No 564220), then 2×10⁵ cells werecentrifuged at 480 xg for 5 min and washed with PBS. Surface stainingfor CD19 (BV650 anti human, BioLegend #302238) was performed accordingto the supplier's indications. Cells were washed once with 150 μl/wellof PBS and resuspended in 150 μl/well of PBS and analyzed using BD FACSFortessa.

In vitro cell binding assays verified that all CD19-CD28 agonisticantibodies bind to human CD19 as well as human CD28 on cells in aconcentration dependent manner (FIGS. 44A and 44B). As expected, nobinding was detected with the anti-DP47 IgG, indicating that thedetection of binding is due to specific CD28 and CD19 binding by therespective targeting moieties. The EC₅₀ values for the binding to CD28are shown in Table 42 and the EC₅₀ values for the binding to CD19 areshown in Table 43.

TABLE 42 EC₅₀ values of CD19-CD28 agonistic antibodies for binding toCD28 ID Molecules EC₅₀ (nM) P1AF0175 CD19-CD28 v8 232 P1AF0377 CD19-CD28v11 150.4 P1AE9040 CD19-CD28 v15 15.55 P1AF0378 CD19-CD28 v27 10.75P1AE8002 CD19-CD28 v29 6.646

TABLE 43 EC₅₀ values of CD19-CD28 agonistic antibodies for binding toCD19 ID Molecules EC₅₀ (nM) P1AF0175 CD19-CD28 v8 0.2324 P1AF0377CD19-CD28 v11 0.3631 P1AE9040 CD19-CD28 v15 0.416 P1AF0378 CD19-CD28 v270.368 P1AE8002 CD19-CD28 v29 0.2742

Example 21

In Vitro Functional Characterization of Bispecific CD28 AgonisticAntigen Binding Molecules Targeting CD19 or CD79b

Several cell-based in vitro assays were performed with primary humanPBMCs to evaluate the activity of CD28(SA) and bispecific CD19 orCD79b-targeted CD28 antigen binding molecules in the presence andabsence of TCR signals provided by T-cell bispecific-(TCB) antibodies.T-cell proliferation, cytokine secretion, and tumor cell killing asdetermined by flow cytometry, cytokine ELISA, and live cell imaging wereobtained as read-outs.

PBMC Isolation

Peripheral blood mononuclear cells (PBMCs) were prepared by densitygradient centrifugation from enriched lymphocyte preparations ofheparinized blood obtained from a Buffy Coat (Blutspende Zurich). 25 mlof blood (diluted 1:2 in PBS) were layered over 15 ml lymphoprep(STEMCELL technologies, Cat No 07851) and centrifuged at roomtemperature for min at 845 xg without brake. The PBMC-containinginterphase was collected in 50 ml tubes with a 10 ml pipette. The cellswere washed with PBS and centrifuged 5 min at 611 xg. The supernatantwas discarded, the pellet re-suspended in 50 ml PBS and centrifuged for5 min at 304 xg. The washing step was repeated, centrifuging at 171 xg.The cells were re-suspended in RPMI 1640 Glutamax (containing 5% humanserum, sodium pyruvate, NEAA, 50 μM 2-mercaptoethanol,Penicillin/Streptomycin) and processed for further functional analysisaccording to the respective assay protocol.

In Vitro Functional Characterization of CD19-CD28 and CD79b-CD28Molecules Based on IL-2 Reporter Assay

To assess the ability of CD19-CD28 and CD79b-CD28 to supportTCB-mediated T cell activation, IL-2 reporter cells (Promega, Ca NoJ1651) served as effector cells (Jurkat T cell line that expresses aluciferase reporter driven by the IL-2 promoter) and Nalm6, RCK8, WSUDLCL2 and Z138 served as tumor targets. 2×10⁴ tumor target cells wereincubated in white flat-bottom 96-well plates for 6 h at 37° C. with 10⁵IL-2 reporter cells (E:T 5:1) in presence of suboptimal CD20-TCB(P1AD4071) concentrations (10 nM for Nalm6 or 0.05 nM for RCK8, WSUDLCL2 and Z138) alone or in combination with increasing concentrationsof the CD19-CD28 (or CD79b-CD28) constructs (0.2 pM-10 nM). Prior to themeasurement, plates were incubated at room temperature for 15 min, andthen 100 ul of substrate (ONE-Glo solution, Promega, Cat No E6120) wasadded to the cells. After 10 min of incubation at room temperature inthe dark, Luminescence (counts/sec) was measured with a Tecan Spark 10M.

In Vitro Functional Characterization of CD19-CD28 Based on PBMC-IsolatedT Cell Activation

To assess the ability of CD19-CD28 to support TCB-mediated T cellactivation, pan T cells were used as effector cells and isolated fromPBMCs by MACS, using the Pan T Cell Isolation Kit (Miltenyi Biotec, CatNo 130-096-535) according to the manufacturer's instructions and Nalm6,RCK8, WSU DLCL2 and Z138 served as tumor targets. 2×10⁴ tumor targetcells were incubated in flat-bottom 96-well plates for 48 h at 37° C.with 10⁵ pan T cells (E:T 5:1) in presence of suboptimal CD20-TCB(P1AD4071) concentrations (10 nM for Nalm6 or 0.05 nM for RCK8, WSUDLCL2 and Z138) alone or in combination with increasing concentrationsof the CD19-CD28 constructs (0.2 pM-10 nM). T cell activation wasassessed via flow cytometry. Briefly, cells were centrifuged at 480 xgfor 5 min and washed with PBS. Surface staining for CD8 (BV421 antihuman, BioLegend #301036), CD4 (PE-Cy7 anti human, BioLegend #344611),CD25 (BV605 anti human, BioLegend #302632), CD69 (PE anti human,BioLegend #310906) was performed according to the supplier'sindications. Cells were washed once with 150 ul/well of PBS andresuspended in 150 ul/well of PBS and analyzed using BD FACS Fortessa.

Cytokine Release Assessment

To assess the ability of CD19-CD28 to trigger cytokine release inpresence of TCR signaling, 5×10⁵ PBMCs were incubated in U-bottom96-well plates for 48 h at 37° C. in presence of CD19-CD28 (1 nM) andsuboptimal CD20-TCB (P1AD4071) concentration (0.4 pM). To assess theability of CD19-CD28 to trigger cytokine release in absence of TCRsignaling, 5×10⁵ PBMCs were incubated in U-bottom 96-well plates for 48h at 37° C. in presence of increasing concentrations of the CD19-CD28constructs (0.08 nM-100 nM). Cytokine release was assessed via Multiplexassay. 50 μl/well of supernatant was screened for G-CSF, GM-CSF, IFN-γ,IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p′70), IL-13,IL-17, MCP-1 (MCAF), MIP-1β and TNF-α secretion using the Bio-Plex ProHuman Cytokine 17-plex Assay (Bio-rad, Cat No m5000031yv) according tothe supplier's indications.

CD19-CD28 Enhances CD20-TCB Mediated T Cell Activation on Various B CellLines

To assess the ability of CD19-CD28 antibodies to enhance CD20-TCBmediated effector function, T cell activation in TCB combination wasassessed. To this end, IL-2 reporter Jurkat cells were co-cultured withfour different CD19-expressing cell lines (Nalm6, WSU DLCL2, Z138, RCK8)for 6 h in presence of increasing concentrations of CD19-CD28 v15(P1AE9040) and fixed, limiting concentration of CD20-TCB (P1AD4071).CD20 TCB is an anti-CD20/anti-CD3 bispecific antibody in a 2+1 format asdescribed in Example 1 of WO 2016/020309 A1. As depicted in FIGS. 45A to45D, CD19-CD28 increases CD20-TCB mediated effector function in presenceof all four B cell lines in a concentration dependent manner.

Affinity-Reduced CD28 Binder Variants are Functional In Vitro in aCD19-CD28 Bispecific Format

The original CD28(SA) clone has an affinity of K_(D)=1 nM. High affinityantibody clones harbor the risk to be subject to peripheral sinkeffects, especially if the target is highly expressed in peripheralblood, as is the case for CD28. In order to (i) reduce peripheral sinkeffects, and (ii) reduce the risk of peripheral T cell activationthrough off-tumor binding of targ. CD28 agonists to T cells, wegenerated a series of 31 CD28 clones with reduced affinities byintroducing point mutations in the CDRs (see Example 1.1). Candidateclones were selected as previously described. 5 molecules with differentCD28 affinities were generated in the CD19-targeted bispecific format:CD19-CD28 v8 (P1AF0175), CD19-CD28 v11 (P1AF0377), CD19-CD28 v15(P1AE9040), CD19-CD28 v27 (P1AF0378) and CD19-CD28 v29 (P1AE8002). FIGS.44A and 44B show that all generated CD19-CD28 molecules bind to humanCD19 as well as CD28, and binding intensities correlate with binderaffinities.

To assess whether affinity-reduced CD28 clone variants were functionaland able to support TCB-mediated effector functions, we assessed T cellactivation in TCB combination. To this end, IL-2 reporter Jurkat cellswere co-cultured with CD19-expressing Nalm6 cells for 6 h in presence ofincreasing concentrations of CD19-CD28 and fixed, limiting concentrationof CD20-TCB. As depicted in FIG. 46 , all variants of the CD28 binderswere functional and able to increase TCB-mediated T cell activation in aconcentration dependent manner. Pan T cells were used as effector cellsand isolated from PBMCs by MACS, using the Pan T Cell Isolation Kit(Miltenyi Biotec) according to the manufacturer's instructions.

CD19-CD28 does not Activate PBMC T Cells in Absence of TCR Signals

To confirm that CD19-CD28 constructs are inactive without a TCR signalsuch as the one provided by CD20-TCB, we assessed the activation statusof PBMC-derived T cells after co-culture with CD20-expressing targetcells (Nalm6) and CD19-CD28 in absence or presence of CD20-TCB. Asdepicted in FIG. 47 , CD19-CD28 enhances CD20-TCB mediated CD69expression in PBMC T cells in a dose-dependent manner, while in absenceof TCB, CD19-CD28 does not lead to T cell activation, even at highconcentrations (100 nM). This finding is confirmed by our observationthat CD19-CD28 did not lead to cytokine release in PBMC derived T cellsafter co-incubation with CD19-CD28 v8 or CD19-CD28 v15 in presence orabsence of CD20-TCB (FIGS. 48A to 48D). In conclusion, these dataconfirm that CD19-CD28 is not a superagonistic antibody but requires aTCR signal, i.e. provided by a TCB, to enhance T cell activation.

CD79b-CD28 Enhances CD20-TCB-Mediated T Cell Activation

In addition to CD19-targeted CD28 agonistic antibodies, we alsogenerated CD79b-targeted CD28 antibodies to assess their ability toenhance CD20-TCB mediated T cell activation. For this, the CD79bpositive B cell line Z138 was used. As shown in FIG. 49A, CD79b-CD28binds to Z138. Further, CD79b-CD28 was able to enhance CD20-TCB-mediatedIL-2 production of IL-2 Jurkat cells when incubated with Z138 inpresence of 0.05 nM CD20-TCB (FIG. 49B).

Example 22

In Vivo Functional Characterization of Bispecific Antigen BindingMolecules Targeting CD28 and CD19

Efficacy Study with CD19-CD28 Bispecific Antigen Binding Molecules withDifferent CD28 Variants in NALM6 Xenograft in Humanized Mice

The efficacy study described in here was aimed to evaluate which CD28variant in the CD19-CD28 bispecific antigen binding molecule will leadto stronger tumor growth inhibition in monotherapy in a CD19-positivehuman lymphoma model in fully humanized NSG mice.

Human NALM6 cells (B cell precursor leukemia) were originally obtainedfrom ATCC and after expansion deposited in the Glycart internal cellbank. Cells were cultured in RPMI containing 10% FCS and 1× Glutamax.The cells were cultured at 37° C. in a water-saturated atmosphere at 5%CO₂. 50 microliters cell suspension (1×10⁶ NALM6 cells) mixed with 50microliters Matrigel were injected subcutaneously in the flank ofanaesthetized mice with a 22G to 30G needle.

Female NSG mice, age 4-5 weeks at start of the experiment (JacksonLaboratory) were maintained under specific-pathogen-free condition withdaily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (ZH225-17). After arrival,animals were maintained for one week to get accustomed to the newenvironment and for observation. Continuous health monitoring wascarried out on a regular basis.

Female NSG mice were injected i.p. with 15 mg/kg of Busulfan followedone day later by an i.v. injection of 1×10⁵ human hematopoietic stemcells isolated from cord blood. At week 14-16 after stem cell injectionmice were bled sublingual and blood was analyzed by flow cytometry forsuccessful humanization. Efficiently engrafted mice were randomizedaccording to their human T cell frequencies into the different treatmentgroups. At that time, mice were injected with tumor cells s.c. asdescribed (FIG. 50 ) and treated with the compounds or Histidine buffer(Vehicle) when tumor size reached appr. 150 mm³ (day18). All mice wereinjected i.v. with 200 μl of the appropriate solution. To obtain theproper amount of compounds per 200 the stock solutions (Table 44) werediluted with Histidine buffer when necessary.

TABLE 4 Compositions used in the in vivo experiment ConcentrationCompound Formulation buffer (mg/mL) CD19-CD28 20 mM Histidine, 5.63(=stock (variants) 140 mM NaCl, solution) pH 6.0 CD19-CD28 20 mMHistidine, 3.53 (=stock (variant 8) 140 mM NaCl, solution) pH 6.0

Tumor growth was measured twice weekly using a caliper and tumor volumewas calculated as followed:

Tv: (W ²/2)×L (W: Width, L: Length)

The study was terminated at day 53. FIG. 51A shows the tumor growthkinetics (Mean, +SEM) and FIGS. 51B to 51D show the individual tumorgrowth kinetics per group and mouse. As described here, CD19-CD28variant 8, as a single agent induced stronger tumor growth inhibition ascompared to CD19-CD28 variant 15. Vehicle animal had to be sacrificedearlier due to the formation of metastasis upon s.c. tumor cellinjection. The monotherapeutic effect in humanized mice can be explainedby the boosting of Allo-reactive human T cells, that can be seen as asurrogate for neo-antigen recognition, in the mouse system.

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1.-63. (canceled)
 64. A polynucleotide or set of polynucleotidesencoding a bispecific agonistic CD28 antigen binding moleculecharacterized by monovalent binding to CD28, wherein said bispecificagonistic CD28 antigen binding molecule comprises (a) one antigenbinding domain capable of specific binding to CD28, wherein the antigenbinding domain capable of specific binding to CD28 comprises a heavychain variable region (VHCD28) comprising a CDR-H1 of SEQ ID NO: 489, aCDR-H2 of SEQ ID NO: 490, and a CDR-H3 of SEQ ID NO:38 (SHYGX5DX6NFDV)wherein X5 is L and X6 is F, and a light chain variable region (VLCD28)comprising a CDR-L1 of SEQ ID NO: 492, a CDR-L2 of SEQ ID NO: 493 and aCDR-L3 of SEQ ID NO: 494; (b) at least one antigen binding domaincapable of specific binding to a tumor-associated antigen, and (c) an Fcdomain composed of a first and a second subunit capable of stableassociation comprising one or more amino acid substitution that reducesthe binding affinity of the antigen binding molecule to an Fc receptorand/or effector function compared to a native IgG1 Fc domain.
 65. Thepolynucleotide or set of polynucleotides of claim 64, wherein the Fcdomain is of human IgG1 subclass and comprises the amino acid mutationsL234A, L235A and P329G, as numbered according to Kabat EU index.
 66. Thepolynucleotide or set of polynucleotides of claim 64, wherein theantigen binding domain capable of specific binding to CD28 comprises aheavy chain variable region (VHCD28) comprising the amino acid sequenceof SEQ ID NO:47 and a light chain variable region (VLCD28) comprisingthe amino acid sequence of SEQ ID NO:54.
 67. The polynucleotide or setof polynucleotides of claim 64, wherein the tumor-associated antigen isselected from the group consisting of Fibroblast Activation Protein(FAP), Carcinoembryonic Antigen (CEA), Folate receptor alpha (FolR1),Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), EpidermalGrowth Factor Receptor (EGFR), human epidermal growth factor receptor 2(HER2), p95HER2, epithelial cell adhesion molecule (EpCAM), HER3, CD30,TPBG (5T4), CD19, CD79b, CD20, CD22, CD37, CD38, BCMA and GPRC5D. 68.The polynucleotide or set of polynucleotides of claim 64, wherein theantigen binding domain capable of specific binding to a tumor-associatedantigen is an antigen binding domain capable of specific binding toCarcinoembryonic Antigen (CEA).
 69. The polynucleotide or set ofpolynucleotides of claim 68, wherein the antigen binding domain capableof specific binding to CEA comprises (i) a heavy chain variable region(V H CEA) comprising a CDR-H1 comprising the amino acid sequence of SEQID NO:180, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:181,and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:182, and alight chain variable region (V L CEA) comprising a CDR-L1 comprising theamino acid sequence of SEQ ID NO:183, a CDR-L2 comprising the amino acidsequence of SEQ ID NO:184, and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:185; or (ii) a heavy chain variable region (V HCEA) comprising a CDR-H1 comprising the amino acid sequence of SEQ IDNO:127, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:128,and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:129, and alight chain variable region (V L CEA) comprising a CDR-L1 comprising theamino acid sequence of SEQ ID NO:130, a CDR-L2 comprising the amino acidsequence of SEQ ID NO:131, and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:132, (iii) a heavy chain variable region (V H CEA)comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:507,a CDR-H2 comprising the amino acid sequence of SEQ ID NO:508, and aCDR-H3 comprising the amino acid sequence of SEQ ID NO:509, and a lightchain variable region (V L CEA) comprising a CDR-L1 comprising the aminoacid sequence of SEQ ID NO:510, a CDR-L2 comprising the amino acidsequence of SEQ ID NO:511, and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:512.
 70. The polynucleotide or set ofpolynucleotides of claim 68, wherein the antigen binding domain capableof specific binding to CEA comprises a heavy chain variable region (V HCEA) comprising the amino acid sequence of SEQ ID NO:186, and a lightchain variable region (V L CEA) comprising the amino acid sequence ofSEQ ID NO:187.
 71. The polynucleotide or set of polynucleotides of claim68, wherein the antigen binding domain capable of specific binding toCEA comprises (a) a heavy chain variable region (V H CEA) comprising theamino acid sequence of SEQ ID NO:194 and a light chain variable region(V L CEA) comprising the amino acid sequence of SEQ ID NO:195, or (b) aheavy chain variable region (V H CEA) comprising the amino acid sequenceof SEQ ID NO:196 and a light chain variable region (V L CEA) comprisingthe amino acid sequence of SEQ ID NO:197, or (c) a heavy chain variableregion (V H CEA) comprising the amino acid sequence of SEQ ID NO:198 anda light chain variable region (V L CEA) comprising the amino acidsequence of SEQ ID NO:199, or (d) a heavy chain variable region (V HCEA) comprising the amino acid sequence of SEQ ID NO:200 and a lightchain variable region (V L CEA) comprising the amino acid sequence ofSEQ ID NO:201, or (e) a heavy chain variable region (V H CEA) comprisingthe amino acid sequence of SEQ ID NO:202 and a light chain variableregion (V L CEA) comprising the amino acid sequence of SEQ ID NO:203, or(f) a heavy chain variable region (V H CEA) comprising the amino acidsequence of SEQ ID NO:204 and a light chain variable region (V L CEA)comprising the amino acid sequence of SEQ ID NO:205, or (g) a heavychain variable region (V H CEA) comprising the amino acid sequence ofSEQ ID NO:206 and a light chain variable region (V L CEA) comprising theamino acid sequence of SEQ ID NO:207, or (h) a heavy chain variableregion (V H CEA) comprising the amino acid sequence of SEQ ID NO:208 anda light chain variable region (V L CEA) comprising the amino acidsequence of SEQ ID NO:209, or (i) a heavy chain variable region (V HCEA) comprising the amino acid sequence of SEQ ID NO:210 and a lightchain variable region (V L CEA) comprising the amino acid sequence ofSEQ ID NO:211, or (j) a heavy chain variable region (V H CEA) comprisingthe amino acid sequence of SEQ ID NO:212 and a light chain variableregion (V L CEA) comprising the amino acid sequence of SEQ ID NO:213.72. The polynucleotide or set of polynucleotides of claim 68, whereinthe antigen binding domain capable of specific binding to CEA comprisesa heavy chain variable region (V H CEA) comprising the amino acidsequence of SEQ ID NO:200 and a light chain variable region (V L CEA)comprising the amino acid sequence of SEQ ID NO:201.
 73. Thepolynucleotide or set of polynucleotides of claim 64, wherein theantigen binding domain capable of specific binding to a tumor-associatedantigen is an antigen binding domain capable of specific binding toFibroblast Activation Protein (FAP).
 74. The polynucleotide or set ofpolynucleotides of claim 73, wherein the antigen binding domain capableof specific binding to FAP comprises: (a) a heavy chain variable region(V H FAP) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:12, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:13, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:14, and a light chain variable region (V L FAP) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:15, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:16, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:17, or (b) a heavy chainvariable region (V H FAP) comprising (i) CDR-H1 comprising the aminoacid sequence of SEQ ID NO:4, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:5, and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO:6, and a light chain variable region (V L FAP)comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:7, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9.
 75. Thepolynucleotide or set of polynucleotides of claim 73, wherein theantigen binding domain capable of specific binding to FAP comprises aheavy chain variable region (V H FAP) comprising the amino acid sequenceof SEQ ID NO:18 and a light chain variable region (V L FAP) comprisingthe amino acid sequence of SEQ ID NO:19.
 76. The polynucleotide or setof polynucleotides of claim 64, wherein the antigen binding domaincapable of specific binding to a tumor-associated antigen is an antigenbinding domain capable of specific binding to epithelial cell adhesionmolecule (EpCAM).
 77. The polynucleotide or set of polynucleotides ofclaim 76, wherein the antigen binding domain capable of specific bindingto EpCAM comprises a heavy chain variable region (V H EpCAM) comprising(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:515, (ii)CDR-H2 comprising the amino acid sequence of SEQ ID NO:516, and (iii)CDR-H3 comprising the amino acid sequence of SEQ ID NO:517, and a lightchain variable region (V L EpCAM) comprising (iv) CDR-L1 comprising theamino acid sequence of SEQ ID NO:518, (v) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:519, and (vi) CDR-L3 comprising the aminoacid sequence of SEQ ID NO:520.
 78. The polynucleotide or set ofpolynucleotides of claim 76, wherein the antigen binding domain capableof specific binding to EpCAM comprises a heavy chain variable region (VH EpCAM) comprising the amino acid sequence of SEQ ID NO:521 and a lightchain variable region (V L EpCAM) comprising the amino acid sequence ofSEQ ID NO:522.
 79. The polynucleotide or set of polynucleotides of claim64, wherein the antigen binding domain capable of specific binding to atumor-associated antigen is an antigen binding domain capable ofspecific binding to HER3.
 80. The polynucleotide or set ofpolynucleotides of claim 79, wherein the antigen binding domain capableof specific binding to HER3 comprises a heavy chain variable region (V HHER3) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ IDNO:523, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:524,and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:525,and a light chain variable region (V L HER3) comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:526, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:527, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:528.
 81. Thepolynucleotide or set of polynucleotides of claim 79, wherein theantigen binding domain capable of specific binding to HER3 comprises aheavy chain variable region (V H HER3) comprising the amino acidsequence of SEQ ID NO:529 and a light chain variable region (V L HER3)comprising the amino acid sequence of SEQ ID NO:530.
 82. Thepolynucleotide or set of polynucleotides of claim 64, wherein theantigen binding domain capable of specific binding to a tumor-associatedantigen is an antigen binding domain capable of specific binding toCD30.
 83. The polynucleotide or set of polynucleotides of claim 82,wherein the antigen binding domain capable of specific binding to CD30comprises a heavy chain variable region (V H CD30) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:531, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:532, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:533, and a light chainvariable region (V L CD30) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:534, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:535, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:536.
 84. The polynucleotide or set ofpolynucleotides of claim 82, wherein the antigen binding domain capableof specific binding to CD30 comprises a heavy chain variable region (V HCD30) comprising the amino acid sequence of SEQ ID NO:537 and a lightchain variable region (V L CD30) comprising the amino acid sequence ofSEQ ID NO:538.
 85. The polynucleotide or set of polynucleotides of claim64, wherein the antigen binding domain capable of specific binding to atumor-associated antigen is an antigen binding domain capable ofspecific binding to TBPG.
 86. The polynucleotide or set ofpolynucleotides of claim 85, wherein the antigen binding domain capableof specific binding to TBPG comprises a heavy chain variable region (V HTBPG) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ IDNO:539, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:540,and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:541,and a light chain variable region (V L TBPG) comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:542, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:543, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:544.
 87. Thepolynucleotide or set of polynucleotides of claim 85, wherein theantigen binding domain capable of specific binding to TBPG comprises aheavy chain variable region (V H TBPG) comprising the amino acidsequence of SEQ ID NO:545 and a light chain variable region (V L TBPG)comprising the amino acid sequence of SEQ ID NO:546.
 88. Thepolynucleotide or set of polynucleotides of claim 64, wherein thetumor-associated antigen is a Multiple Myeloma (MM) cell surface antigenselected from the group consisting of CD38, BCMA and GPRC5D.
 89. Thepolynucleotide or set of polynucleotides of claim 88, wherein theantigen binding domain capable of specific binding to a tumor-associatedantigen is an antigen binding domain capable of specific binding toGPRC5D.
 90. The polynucleotide or set of polynucleotides of claim 88,wherein the antigen binding domain capable of specific binding to GPRC5Dcomprises (a) a heavy chain variable region (V H GPRC5D) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:563, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:564, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:565, and a light chainvariable region (V L GPRC5D) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:566, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:567, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:568, or (b) a heavy chain variable region (V HGPRC5D) comprising (i) CDR-H1 comprising the amino acid sequence of SEQID NO:579, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:580, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:581, and a light chain variable region (V L GPRC5D) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:582, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:583, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:584.
 91. Thepolynucleotide or set of polynucleotides of claim 88, wherein theantigen binding domain capable of specific binding to GPRC5D comprises aheavy chain variable region (V H GPRC5D) comprising the amino acidsequence of SEQ ID NO:569 and a light chain variable region (V L GPRC5D)comprising the amino acid sequence of SEQ ID NO:570.
 92. Thepolynucleotide or set of polynucleotides of claim 64, wherein theantigen binding domain capable of specific binding to a tumor-associatedantigen is an antigen binding domain capable of specific binding toCD38.
 93. The polynucleotide or set of polynucleotides of claim 92,wherein the antigen binding domain capable of specific binding to CD38comprises a heavy chain variable region (VHCD38) comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:547, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:548, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:549, and a light chainvariable region (V L CD38) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:550, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:551, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:552.
 94. The polynucleotide or set ofpolynucleotides of claim 92, wherein the antigen binding domain capableof specific binding to CD38 comprises a heavy chain variable region(VHCD38) comprising the amino acid sequence of SEQ ID NO:553 and a lightchain variable region (V_(L)CD38) comprising the amino acid sequence ofSEQ ID NO:554.
 95. The polynucleotide or set of polynucleotides of claim64, wherein the antigen binding domain capable of specific binding to atumor-associated antigen is an antigen binding domain capable ofspecific binding to BCMA.
 96. The polynucleotide or set ofpolynucleotides of claim 95, wherein the antigen binding domain capableof specific binding to BCMA comprises a heavy chain variable region (V HBCMA) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ IDNO:555, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:556,and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:557,and a light chain variable region (V L BCMA) comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:558, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:559, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:560.
 97. Thepolynucleotide or set of polynucleotides of claim 32, wherein theantigen binding domain capable of specific binding to BCMA comprises aheavy chain variable region (V H BCMA) comprising the amino acidsequence of SEQ ID NO:561 and a light chain variable region (V L BCMA)comprising the amino acid sequence of SEQ ID NO:562.
 98. Thepolynucleotide or set of polynucleotides of claim 64, wherein thetumor-associated antigen is a B cell surface antigen selected from thegroup consisting of CD19, CD79b, CD20, CD22 and CD37.
 99. Thepolynucleotide or set of polynucleotides of claim 64, wherein theantigen binding domain capable of specific binding to a tumor-associatedantigen is an antigen binding domain capable of specific binding toCD19.
 100. The polynucleotide or set of polynucleotides of claim 99,wherein the antigen binding domain capable of specific binding to CD19comprises: (a) a heavy chain variable region (VHCD19) comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:406, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:407, and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:408, and a light chainvariable region (VLCD19) comprising (iv) CDR-L1 comprising the aminoacid sequence of SEQ ID NO:409, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO:410, and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO:411, or (b) a heavy chain variable region (VHCD19)comprising (i) CDR-H1 comprising the amino acid sequence of SEQ IDNO:414, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:415,and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:416,and a light chain variable region (VLCD19) comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:417, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:418, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:419.
 101. Thepolynucleotide or set of polynucleotides of claim 99, wherein theantigen binding domain capable of specific binding to CD19 comprises (a)a heavy chain variable region (VHCD19) comprising the amino acidsequence of SEQ ID NO:412, and a light chain variable region (VLCD19)comprising the amino acid sequence of SEQ ID NO:413, or (b) a heavychain variable region (VHCD19) comprising the amino acid sequence of SEQID NO:420, and a light chain variable region (VLCD19) comprising theamino acid sequence of SEQ ID NO:421.
 102. The polynucleotide or set ofpolynucleotides of claim 99, wherein the antigen binding domain capableof specific binding to CD19 comprises a heavy chain variable region(VHCD19) comprising the amino acid sequence of SEQ ID NO:412 and a lightchain variable region (VLCD19) comprising the amino acid sequence of SEQID NO:413.
 103. The polynucleotide or set of polynucleotides of claim64, wherein the antigen binding domain capable of specific binding to atumor-associated antigen is an antigen binding domain capable ofspecific binding to CD79b.
 104. The polynucleotide or set ofpolynucleotides of claim 103, wherein the antigen binding domain capableof specific binding to CD79b comprises a heavy chain variable region(VHCD79b) comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:422, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:423, and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:424, and a light chain variable region (V L CD79b) comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:425, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:426, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:427.
 105. Thepolynucleotide or set of polynucleotides of claim 103, wherein theantigen binding domain capable of binding to CD79b comprises a heavychain variable region (V H CD79b) comprising the amino acid sequence ofSEQ ID NO:428, and a light chain variable region (VLCD79b) comprisingthe amino acid sequence of SEQ ID NO:429.
 106. The polynucleotide or setof polynucleotides of claim 64, wherein the bispecific agonistic CD28antigen binding molecule comprises: (a) one Fab fragment capable ofspecific binding to CD28, (b) one crossFab fragment capable of specificbinding to a tumor-associated antigen, and (c) a Fc domain composed of afirst and a second subunit capable of stable association comprising oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or effector functioncompared to a native IgG1 Fc domain.
 107. The polynucleotide or set ofpolynucleotides of claim 64, wherein the bispecific agonistic CD28antigen binding molecule comprises: (a) a first Fab fragment capable ofspecific binding to CD28, (b) a second Fab fragment capable of specificbinding to a tumor-associated antigen, and (c) a Fc domain composed of afirst and a second subunit capable of stable association comprising oneor more amino acid substitution that reduces the binding affinity of theantigen binding molecule to an Fc receptor and/or effector functioncompared to a native IgG1 Fc domain, wherein the first Fab fragmentcapable of specific binding to CD28 is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab fragment capable of specific binding to a tumor-associated antigen,which is in turn fused at its C-terminus to the N-terminus of one of theFc domain subunits.
 108. The polynucleotide or set of polynucleotides ofclaim 64, wherein the bispecific agonistic CD28 antigen binding moleculecomprises: (a) a first Fab fragment capable of specific binding to CD28,(b) a second and a third Fab fragment capable of specific binding to atumor-associated antigen, and (c) a Fc domain composed of a first and asecond subunit capable of stable association comprising one or moreamino acid substitution that reduces the binding affinity of the antigenbinding molecule to an Fc receptor and/or effector function compared toa native IgG1 Fc domain, wherein the first Fab fragment capable ofspecific binding to CD28 is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second Fabfragment capable of specific binding to a tumor-associated antigen,which is in turn fused at its C-terminus to the N-terminus of the firstFc domain subunit, and the third Fab fragment capable of specificbinding to a tumor-associated antigen is fused at the C-terminus of theFab heavy chain to the N-terminus of the second Fc domain subunit. 109.The polynucleotide or set of polynucleotides of claim 64, wherein thebispecific agonistic CD28 antigen binding molecule comprises: (a) oneantigen binding domain capable of specific binding to CD28, wherein theantigen binding domain capable of specific binding to CD28 comprises aheavy chain variable region (V H CD28) comprising the amino acidsequence of SEQ ID NO:47 and a light chain variable region (VLCD28)comprising the amino acid sequence of SEQ ID NO:54, (b) at least oneantigen binding domain capable of specific binding to CD19, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation, wherein the Fc domain is of human IgG1 subclass andcomprises the amino acid mutations L234A, L235A and P329G, numberingaccording to Kabat EU index.
 110. The polynucleotide or set ofpolynucleotides of claim 109, wherein the antigen binding domain capableof specific binding to CD19 comprises a heavy chain variable region(VHCD19) comprising the amino acid sequence of SEQ ID NO:412 and a lightchain variable region (VLCD19) comprising the amino acid sequence of SEQID NO:413.
 111. A polynucleotide or set of polynucleotides encoding abispecific agonistic CD28 antigen binding molecule comprising a firstlight chain comprising the amino acid sequence of SEQ ID NO:122, a firstheavy chain comprising the amino acid sequence of SEQ ID NO:114, asecond heavy chain comprising the amino acid sequence of SEQ ID NO:430and a second light chain comprising the amino acid sequence of SEQ IDNO:431.
 112. A vector comprising the polynucleotide or set ofpolynucleotides of claim
 64. 113. A cell comprising the vector of claim112.
 114. A method of producing bispecific agonistic CD28 antigenbinding molecule comprising culturing the host cell of claim 113 underconditions suitable for the expression of the bispecific agonistic CD28antigen binding molecule, and recovering the bispecific agonistic CD28antigen binding molecule.